Identification of potent and selective small-molecule inhibitors of caspase-3 through the use of extended tethering and structure-based drug design

Article abstract of DOI:10.1021/jm020230j

The design, synthesis, and in vitro activities of a series of potent and selective small-molecule inhibitors of caspase-3 are described. From extended tethering, a salicylic acid fragment was identified as having binding affinity for the S₄ pocket of caspase-3. X-ray crystallography and molecular modeling of the initial tethering hit resulted in the synthesis of 4, which reversibly inhibited caspase-3 with a K_i = 40 nM. Further optimization led to the identification of a series of potent and selective inhibitors with K_i values in the 20-50 nM range. One of the most potent compounds in this series, 66b, inhibited caspase-3 with a K_i = 20 nM and selectivity of 8-500-fold for caspase-3 vs a panel of seven caspases (1, 2, and 4-8). A high-resolution X-ray cocrystal structure of 4 and 66b supports the predicted binding modes of our compounds with caspase-3.

Full text of DOI:10.1021/jm020230j

J . Med. Chem. 2002, 45, 5005-5022
5005
Id en tifica tion of P oten t a n d Selective Sm a ll-Molecu le In h ibitor s of Ca sp a se-3
th r ou gh th e Use of Exten d ed Teth er in g a n d Str u ctu r e-Ba sed Dr u g Design
Ingrid C. Choong,* Willard Lew, Dennis Lee, Phuongly Pham, Matthew T. Burdett, J oni W. Lam,
Christian Wiesmann, Tinh N. Luong, Bruce Fahr, Warren L. DeLano, Robert S. McDowell, Darin A. Allen,
Daniel A. Erlanson, Eric M. Gordon, and Tom O’Brien
Sunesis Pharmaceuticals, Inc., 341 Oyster Point Boulevard, South San Francisco, California 94080
Received May 28, 2002
The design, synthesis, and in vitro activities of a series of potent and selective small-molecule
inhibitors of caspase-3 are described. From extended tethering, a salicylic acid fragment was
identified as having binding affinity for the S4 pocket of caspase-3. X-ray crystallography and
molecular modeling of the initial tethering hit resulted in the synthesis of 4, which reversibly
inhibited caspase-3 with a Ki ) 40 nM. Further optimization led to the identification of a series
of potent and selective inhibitors with Ki values in the 20-50 nM range. One of the most potent
compounds in this series, 66b, inhibited caspase-3 with a Ki ) 20 nM and selectivity of 8-500-
fold for caspase-3 vs a panel of seven caspases (1, 2, and 4-8). A high-resolution X-ray cocrystal
structure of 4 and 66b supports the predicted binding modes of our compounds with caspase-
3.
In tr od u ction
kine pro-IL-1â in monocytes to the active 17 kDa
cytokine IL-1â, a key inflammatory mediator. The
second subfamily is comprised of caspases involved in
mediating programmed cell death or apoptosis. The
upstream members in the apoptotic signaling cascade
are referred to as initiator caspases (caspase-2, -8, -9,
and -10) while downstream members are referred to as
effector caspases (caspase-3, -6, and -7). Initiator caspas-
es are activated in response to proapoptotic signals and
are responsible for activating the effector caspases,
which in turn are responsible for the proteolytic cleav-
age of substrates involved in the final stages of cell
disassembly.5,10 It is worth noting that the exact roles
and functions of individual caspase members in the
apoptotic signaling pathway have not been fully deci-
phered at this time.
Not surprisingly, as a result of their roles in inflam-
mation and apoptosis, the caspases have generated
substantial interest from both the pharmaceutical and
the biotechnology sector as potential targets for drug
discovery. Inflammatory diseases such as rheumatoid
arthritis and diseases involving dysregulated apoptosis
such as myocardial infarction, stroke, traumatic brain
injury, sepsis, Alzheimer’s disease, and Parkinson’s
disease represent a large unmet medical need and could
benefit from potent and selective caspase inhibitors.11-13
Studies involving knockout mice as well as numerous
proof-of-concept animal models have demonstrated that
regulation of caspase activity may be a viable thera-
peutic approach.2,14,15
Cysteine proteases comprise a distinct mechanistic
class of proteolytic enzymes that have been widely
implicated as potential therapeutic targets. To date,
three structural classes of cysteine proteases are known,
(i) the ICE (interleukin-1â converting enzyme) class, (ii)
the papain class (e.g., cathepsins), and (iii) the picor-
novirus 3C-protease class. A number of reviews have
been published.1-3
Of specific interest to us are the caspases (cysteinyl
aspartate specific proteases), which play key roles in
both cytokine maturation and programmed cell death
(apoptosis).4,5 Common to all caspases is the presence
of a catalytic diad comprised of a nucleophilic cysteine
and histidine imidazole ring. All caspases cleave their
respective peptide substrates after an aspartic acid
residue; it is this near absolute requirement for aspartic
acid at the P1 position6 of their substrates that makes
the caspases some of the most specific proteases known.
This specificity can be largely attributed to favorable
hydrogen-bonding interactions between the charged
aspartate residue of the substrate and the three amino
acid residues Arg64, Gln161, and Arg207 in the caspase
active site (caspase-3 numbering adopted from previous
studies).7 Studies using a positional-scanning combina-
torial library have demonstrated that the S4 subsite is
the single most important determinant of specificity
among the caspases.8,9 To date, at least 12 human
caspase members have been identified. On the basis of
their sequence homology, substrate specificity, and
structural similarities, the caspases can be divided into
two major subfamilies. Those related to ICE (caspase-
1, -4, -5, and -13) are involved in inflammation (cytokine
maturation). Caspase-1 was the first caspase member
to be identified and is responsible for the cleavage of
the Asp116-Ala117 bond in the inactive 31 kDa cyto-
In addition to being potential therapeutic agents,
potent and selective caspase inhibitors would be invalu-
able tools in the study and delineation of the roles that
individual caspases play in the complex apoptosis
signaling pathways. The majority of cell-based assays
and animal models studying the neuroprotective effects
of caspase inhibitors make use of peptide-derived in-
hibitors, both reversible and irreversible. Although quite
potent, most reversible peptide inhibitors are not highly
* To whom correspondence should be addressed. Tel: (650)266-3647.
Fax: (650)266-3501. E-mail: ichoong@sunesis.com.
10.1021/jm020230j CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/15/2002

5006 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Choong et al.
F igu r e 1. Initial leads developed from an extended tethering screen.
Sch em e 1. General Scheme for Analogue Synthesis
specific for any one caspase. For example, the tetrapep-
tide inhibitor DEVD-CHO, which contains the caspase-3
consensus cleavage sequence, inhibits caspase-3 with a
Ki value of 0.23 nM but also inhibits caspase-1 with a
Ki value of 18 nM.16 Irreversible inhibitors have been
shown to be effective in a variety of proof-of-concept
pharmacological models. However, because of the fact
that these are irreversible inhibitors, they potentially
lack selectivity and the observed results do not neces-
sarily imply inhibition of caspases only.17
Recently, we reported the use of “extended tethering”,
a fragment-based ligand discovery technology, to iden-
tify a novel pharmacophore that was successfully com-
bined with a known fragment to create a unique
nonpeptidic inhibitor of caspase-3.18 In this strategy, a
known binding fragment of the target, in this case an
aspartyl group, is covalently attached to a binding
pocket in the protein active site. The bound fragment
bears a free thiol group, which is allowed to react
reversibly with a library of disulfide-containing com-
pounds. In doing so, the “extender” probes the disulfide
library for additional binding elements for proximal
binding pockets of the active site. In the case of caspase-
3, conversion of initial tethering hit 1 to a reversible
small-molecule compound resulted in inhibitor 2 with
low micromolar affinity (Ki ) 2.8 µM) (Figure 1).
Rigidification of the acyclic linker with a phenyl linker
resulted in compound 3, which displayed 14-fold in-
crease in binding affinity (Ki ) 0.20 µM).
efforts have produced some of the most specific nonpep-
tidic caspase-3 inhibitors reported to date, which display
nanomolar potencies. In addition, X-ray crystallographic
studies have yielded crystal structures, which support
the predicted binding modes of our compounds with
caspase-3.
Ch em istr y
Syn th etic Str a tegy. Unless otherwise noted, aspart-
yl aldehyde and ketone-based inhibitors were prepared
utilizing previously reported methods (Scheme 1). N-
Fluorenylmethoxycarbonyl(Fmoc)-protected aspartyl al-
dehyde or thiomethyl ketone, prepared from the N-Fmoc-
Asp(Ot-Bu)-OH,isattachedtosupport usinga semicarbazide
linker to provide support-bound semicarbazone.19,20
Linking through the invariant carbonyl is ideal because
it protects the carbonyl from nucleophilic attack as well
as racemization at the R-stereocenter.21,22 Removal of
the Fmoc group under basic conditions, followed by
acylation with Fmoc-protected heterocycles, introduces
a rigid linker, which links the aspartyl group to the S4
binding fragment (e.g., salicylic acid). Removal of the
newly incorporated Fmoc group followed by acylation
with sulfonyl chlorides introduces the S4 binding ele-
ment. Finally, acidic cleavage of the semicarbazone
linker releases the fully functionalized aldehyde/ketone
inhibitors from the support.
Syn th esis of Su bstitu ted Ben zen esu lfon yl Ch lo-
r id es. The synthesis of noncommercially available
substituted benzenesulfonyl chlorides used in the prepa-
ration of compounds in Tables 1 and 5 is shown in
Scheme 2. Starting with an appropriately substituted
In this paper, we describe the optimization of com-
pound 3 through modification of the salicylic acid group,
the active site electrophile, and the linker region. These

Small Molecule Inhibitors of Caspase-3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5007
Sch em e 2. Synthesis of Sulfonyl Chloridesa
a
Reagents and conditions (reactions 1 and 2): (a) 10% Pd/C, H2, EtOH, HCl. (b) NaNO2, SO2, CuCl2, HCl, HOAc. Reagents and conditions
(reaction 3): (a) Boc2O, Et3N, THF, 60 °C. (b) Aqueous NH2OH, EtOH, 80 °C. (c) (i) 2-Ethylhexyl chlororformate, pyridine, THF, 0 °C; (ii)
xylene, reflux. (d) 4.0 N HCl, room temperature. (e) NaNO2, SO2, CuCl2, HCl, HOAc.
Sch em e 3. Synthesis of 2,5-Disubstituted Nicotinic
of 63a ,b was prepared in the same manner as shown
for the substitution of 16 except for the substitution of
5-methyl-2-pyrazinecarboxylate for 6-methyl nicotinate.
Acida
The 2,5-disubstituted thiophene linker 21 was pre-
pared in seven steps starting from commercially avail-
able 2-bromothiophene-5-carboxaldehyde 17 according
to Scheme 4. Aldehyde oxidation with NaClO2/NaH2-
PO4 followed by esterification with tert-butyl alcohol
gave the corresponding t-butyl ester 18. Lithium-
halogen metal exchange with n-butyllithium followed
by trapping with 1-formylpiperidine provided aldehyde
19. Conversion of 19 to the oxime with aqueous hy-
droxylamine followed by reduction with zinc dust gave
primary amine 20. Subsequent Fmoc protection and
ester hydrolysis afforded linker 21. Linker 21 was
converted to the N-methylated linker 22 in one pot using
formaldehyde followed by reduction with triethylsilane.
The 3,5-disubstituted thiophene used in the synthesis
of 68a ,b was prepared in the same manner as shown
for 21 except for the substitution of 3-bromothiophene-
5-carboxaldehyde for 2-bromothiophene-5-carboxalde-
hyde.
a
Reagents and conditions: (a) SOCl2, MeOH. (b) NBS, (Ph-
CO2)2, HOAc. (c) NaN3, DMF, 70 °C. (d) 10% Pd/C, H2, EtOH. (e)
Aqueous LiOH, dioxane. (f) Fmoc-OSu, NaHCO3.
Sch em e 4. Synthesis of 2,5-Disubstituted Thiophene
Linkera
The corresponding 2,4-disubstituted thiophene linker
28 was prepared from aldehyde 23 as outlined in
Scheme 5. Sodium borohydride reduction and tert-
butyldimethylsilyl (TBS) protection of the primary
alcohol gave the silyl ether 24. Treatment of 24 with
n-butyllithium followed by trapping with ethyl chloro-
formate provided ethyl ester 25. Conversion of 25 to
bromide 26 was accomplished by first TBS deprotection
with fluoride ion followed by bromination with CBr4 and
triphenylphosphine. Treatment of 26 with sodium azide
and subsequent catalytic hydrogenation furnished amine
27. Saponification followed by Fmoc protection gave
linker 28.
a
Reagents and conditions: (a) NaClO2. (b) t-BuOH, EDC,
DMAP. (c) n-BuLi, -78 °C and then 1-formylpiperidine. (d)
Aqueous NH2OH. (e) Zn, HOAc. (f) Fmoc-OSu, NaHCO3. (g) TFA.
(h) CH2O, TFA and then triethylsilane.
aniline or nitrobenzene precursor, sulfonyl chlorides
5-9 were obtained by either (i) a nitro group reduction
via catalytic hydrogenation followed by chlorosulfony-
lation (compounds 5 and 6) or (ii) a single chlorosulfo-
nylation step (compounds 7-9). The corresponding
5-oxo-1,2,4-oxadiazole 1223 was prepared in five steps
in good yield from commercially available 3-amino
benzonitrile 10.
Syn th esis of F m oc-P r otected Heter ocyclic Lin k -
er s. Fmoc-protected heterocyclic linkers used in the
synthesis of target compounds shown in Table 3 were
prepared according to Schemes 3-5. Esterification of
commercially available 6-methyl nicotinate 13 followed
by bromination via halogenation with N-bromosuccin-
imide (NBS) gave 14. Bromide displacement with
sodium azide followed by catalytic hydrogenation gave
primary amine 15. Final saponification with aqueous
lithium hydroxide followed by Fmoc protection afforded
16. The 2,5-disubstituted pyrazine used in the synthesis
The corresponding Fmoc-protected pyrimidine, thia-
zole, and isoxazole used in the synthesis of 62a ,b, 64a ,b,
and 65a ,b, respectively, were prepared according to
literature methods.24-29 The disubstituted furan linker
used in the preparation of 70a ,b was synthesized using
the same method as linker 28 except for the substitution
of commercially available 5-chloromethyl-furan-2-car-
boxylic acid ethyl ester for intermediate 26.
Syn th esis of Wa r h ea d Gr ou p s. The thiomethyl
ketone derivatives were prepared as shown in Scheme
6. N-Fmoc-Asp(Ot-Bu)-CH2Br 29, synthesized in three
steps from literature methods,20 was reacted with the

5008 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Sch em e 5. Synthesis of 3,5-Disubstituted Thiophene Linkera
Choong et al.
a
Reagents and conditions: (a) NaBH4, THF. (b) TBSCl, imidazole, CH2Cl2. (c) n-BuLi, THF, -78 °C and then ClCO2Et. (d) TBAF,
HOAc, THF. (e) CBr4, PPh3, THF. (f) NaN3, DMF, 50 °C. (g) 10% Pd/C, H2, EtOH. (h) Aqueous LiOH, dioxane. (i) Fmoc-OSu, NaHCO3.
Sch em e 6. Synthesis of Thiomethyl Ketone Warheadsa
no product. However, treatment with dimethylforma-
mide (DMF) di-tert-butyl acetal31 gave the desired
product 39 in modest yields. Saponification of 39 yielded
the elaborated thiophenecarboxylic acid 40, which was
coupled with the appropriate amino alcohols 41a and
42a 34 using standard EDC and HOBt coupling condi-
tions. Dess-Martin periodinane oxidation of the second-
ary alcohol followed by hydrolysis of the t-butyl ester
afforded the keto-oxazole and keto-benzoxazole ana-
logues 41b and 42b, respectively.
Syn th esis of Ald eh yd e a n d Th iom eth yl Keton e
An a logu es. All compounds bearing either the aldehyde
warhead (Tables 1 and 3) or the methyl ketone warhead
(Tables 3-6) were prepared as shown in Scheme 9.
Standard Fmoc deprotection and amine acylation pro-
tocols were used with the two methods differing only in
the conditions for cleavage of the final product. In the
case of the aldehyde warhead, the aldehyde was re-
leased from support after treatment with a cocktail of
tetrahydrofuran (THF)/HOAc/CH3CHO/trifluoroacetic
acid (TFA) followed by hydrolysis of the tert-butyl ester
with TFA/CH2Cl2/H2O. The ketone analogues were
released from support and hydrolyzed in a one step
treatment with TFA/H2O.
Analogues 47a -c were prepared as shown in Scheme
10. Following removal of the Fmoc group, resin 44a was
acylated with either linker 21 or 22. Removal of the
second Fmoc group followed by treatment with 5-formyl-
2-hydroxy-benzoic acid and NaBH(OAc)3 provided the
reductive amination products. Treatment with TFA
provided 47a ,c. Analogue 47b was prepared in the same
manner as 47a except the reductive amination step was
followed by acylation with acetic anhydride.
a
Reagents and conditions: (a) 2-Chlorobenzenemethanethiol,
THF. (b) Thiourea, EtOH. (c) Aqueous NaOH. (d) Compound 29,
DIEA, THF.
Sch em e 7. Synthesis of Phenylpropyl Ketone
Warheadsa
a
Reagents and conditions: (a) n-BuLi, THF, -78 °C and then
N-Fmoc-Asp(Ot-Bu)-CHO. (b) 10% Pd/C, H2, EtOH. (c) Dess-
Martin periodinane, CH2Cl2.
appropriately substituted 2-chlorobenzene methanethiol
or pyridylmethanethiols30 32a ,b to furnish thiomethyl
ketones 30 or 33a ,b, respectively, which were attached
to support 43 for analogue synthesis.
The phenylpropyl ketone intermediate 35 was syn-
thesized in a three-step sequence as shown in Scheme
7. The anion of 3-phenyl-1-propyne 34 was reacted with
N-Fmoc-Asp(Ot-Bu)-CHO,31 hydrogenated over Pd(0),
and oxidized with Dess-Martin periodinane to afford
35. Efforts to generate the phenylpropyl ketone through
the addition of phenylpropyl Grignard to N-Fmoc-Asp-
(Ot-Bu)-CHO resulted in no reaction. The fully func-
tionalized analogue was prepared by attaching com-
pound 35 to the support through the semicarbazide
linker 43 followed by standard analogue synthesis.
The keto-oxazole and keto-benzoxazole analogues 41b
and 42b were prepared in solution as shown in Scheme
8. The elaborated thiophenecarboxylic acid 40 was
prepared starting from commercially available 36. Es-
terification with diazomethane followed by bromination
with NBS provided bromide 37. Treatment with azide
followed by zinc reduction and acylation with 5-chloro-
sulfonyl-2-hydroxybenzoic acid provided 38. It was
necessary at this point to differentially protect the
salicylic acid as the tert-butyl ester. Standard conditions
using EDC/tert-butyl alcohol or H2SO4/MgSO432 yielded
Resu lts a n d Discu ssion
As previously reported, a novel salicylic acid fragment
that binds in the S4 subsite of caspase-3 was identified
by extended tethering.18 Incorporation of the salicylic
acid fragment onto an acyclic linker containing the
requisite Asp-CHO warhead at P1 provided compound
2, which possessed a Ki value of 2.8 µM for caspase-3
inhibition (Figure 1). Upon examination of the X-ray
crystal structure of the tethered hit 118 bound in the
caspase-3 active site, we reasoned that conformational
restriction of the flexible acyclic linker and, thus, the
relative positions of the salicylic acid and sulfonamide
groups in compound 2 could provide improvements in
binding affinity. To this end, compound 3 was synthe-
sized in which a phenyl ring replaces the four carbon
acyclic linker of compound 2. This substructure was
selected in order to maintain the distance between the
salicylic acid and the aspartic acid warhead while

Small Molecule Inhibitors of Caspase-3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5009
Sch em e 8. Synthesis of Keto-Oxazole Analoguesa
a
Reagents and conditions: (a) TMSCH2N2. (b) NBS, (PhCO2)2. (c) NaN3, AcCN. (d) Zn, HOAc. (e) 5-Chlorosulfonyl-2-hydroxybenzoic
acid, CH2Cl2. (f) DMF di-tert-butyl acetal, 90 °C. (g) Aqueous LiOH, dioxane. (h) Compounds 41a or 42a , EDC, NMM, HOBt, DMF. (i)
Dess-Martin periodinane, CH2Cl2. (j) TFA, CH2Cl2.
Sch em e 9. Solid-Phase Synthesis of Carbonyl Analoguesa
a
Reagents and conditions: (a) Compound 30 or 33a or 33b or 35, HOAc, THF. (b) Piperidine, DMF. (c) N-Fmoc-heterocyclic acid,
PyBOP, DIPEA, DMF. (d) Piperidine, DMF. (e) R2SO2Cl, DIPEA, CH2Cl2. (f) 9:1 TFA/H2O. (g) 5:1:1:0.25 THF/HOAc/CH3CHO/TFA. (h)
1:1:0.1 TFA/CH2Cl2/H2O.
simultaneously providing overall rigidification of the
molecule. Compound 3 exhibited 14-fold increase in
inhibitory potency as compared to 2 with Ki values of
0.20 and 2.8 µM, respectively. Molecular modeling
studies of 3, working from the crystal structure of the
initial tethering hit, suggested that this nonpeptidic
structure was capable of replicating many of the key
hydrogen-bonding interactions formed by peptidic in-
hibitors. However, this inhibitor appeared unable to
preserve a common hydrogen-bonding interaction nor-
mally formed between the main chain N-H of Arg207
and the main chain carbonyl of the P3 residue (Figure
2). Thus, we reasoned that this conserved hydrogen-
bonding interaction might be restored through incor-
poration of a hydrogen bond-accepting moiety in the
phenyl ring of 3. Replacement of the phenyl ring with
a 2,5-disubstituted pyridyl ring provided compound 4,
which exhibited a 4-fold improvement in potency over
3 with a Ki ) 0.05 µM. The fluoromethyl ketone
analogue of 4 was prepared to facilitate cocrystallization
for X-ray crystallography. Through this structure of 4,
we were able to confirm the formation of a hydrogen
bond interaction of the pyridyl ring with Arg207 (Figure
3). With compound 4 as a suitable starting point, we
then proceeded with our optimization strategy.
Str u ctu r e-Activity Str a tegy. With the goal of
improving in vitro potency and obtaining good cellular
activity of compound 4, we systematically examined
three regions of the molecule: the salicylic acid group,
the aspartyl aldehyde warhead, and the linker portion

5010 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Choong et al.
Sch em e 10a
F igu r e 3. X-ray cocrystal structure of an irreversible deriva-
tive of compound 4 bound within the active site of caspase-3.
Hydrogen-bonding interaction of pyridyl nitrogen with Arg207
and other key interactions is shown by dashed yellow lines.
a
Reagents and conditions: (a) Piperidine, DMF. (b) Compound
21 or 22, PyBOP, DIPEA. (c) Piperidine, DMF. (d) 5-Formyl-2-
hydroxy-benzoic acid, NaBH(OAc)3. (e) 9:1 TFA/H2O. (f) Ac2O,
DIPEA.
F igu r e 4. Optimization strategy of pyridine analogue 4.
obtained through incorporation of various heterocycles.
Such linking functionality has the potential to form
various hydrogen-bond interactions in the active site
directing appropriate substituents into the S2 and/or S3
subsites of the enzyme.
P 4 Sa licylic Acid Gr ou p Mod ifica tion s of Com -
p ou n d 4. Truncations of the salicylic acid group were
examined to identify which aspects of this fragment
were critical for enzyme binding (Table 1). Removal of
the 4-hydroxyl group (50) resulted in a modest decrease
in caspase-3 inhibitory activity, Ki ) 0.16 µM. In
contrast, binding affinity was significantly decreased
when the carboxylic acid at the 3-position was removed
(49), Ki ) 2.3 µM. Molecular modeling studies based on
the crystal structure of the tethered fragment suggest
that hydrogen bonds are likely formed between this
important carboxylate and the indole of Trp214 and
perhaps the side chain of Asn208 but not the main chain
of Phe250, as would normally be the case for aspartyl
P4 substituents.
To address possible cell membrane permeability is-
sues, nonacidic pharmacophores such as the methyl
sulfone (51) and methyl sulfonamide (52) were selected
for their potential ability to maintain the important
hydrogen-bond interaction to Trp214. These compounds
however resulted in a loss of inhibitory activity with Ki
values of 1.7 and 1.9 µM for compounds 51 and 52,
respectively. It appears that the only P4 substituents
that can maintain moderate caspase-3 inhibitory activ-
ity (within 10-fold of 4) are those possessing acidic
functionality. Namely, incorporation of known carboxy-
lic acid bioisosteres such as an acylsulfonamide (55),
tetrazole (56), or a 5-oxo-1,2,4-oxadiazole moiety (57)
gave compounds with Ki values of 0.47, 0.44, and 0.40
µM, respectively. Interestingly, the reverse acylsulfona-
mide (54) showed only weak activity with Ki ) 1.4 µM.
Asp a r tyl Ald eh yd e Wa r h ea d An a logu es of Com -
p ou n d 4. On the basis of evidence of a hydrophobic
binding pocket in the S1′ subsite of caspases, several
F igu r e 2. Crystal structures of irreversible fluoromethyl
ketone analogues of (a) peptide AcDVAD and (b) compound 3.
In panel b, note the absence of a hydrogen-bonding group
capable of interacting with the main chain N-H of Arg207.
(Figure 4). An immediate goal was to remove one of the
two negative charges present in the molecule to facili-
tate cell membrane permeability. Specifically, we wanted
to determine whether the carboxylic acid group of the
salicylic acid could be replaced with a neutral surrogate
capable of forming an equivalent set of hydrogen-bond
interactions. We also investigated alternative warheads
containing activated ketones as a means of avoiding the
potential metabolic liability of the starting aldehyde.
The use of activated ketones with cysteine proteases is
well-precedented35 and can permit formation of ad-
ditional binding interactions in the S1′ site of the
enzyme. Finally, the linker portion of the molecule was
examined to see if improvements in potency could be

Small Molecule Inhibitors of Caspase-3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5011
Ta ble 1. Caspase-3 Inhibition by Substituted P4 Aryl
Ta ble 2. Caspase-3 Inhibition by P1 Aspartyl Ketone
Sulfonamides on a Pyridine Scaffold
Derivatives on a Pyridine Scaffold
Ta ble 3. Caspase-3 Inhibition by Heterocyclic Derivatives: P1
Aspartyl Aldehyde vs Aryl Thiomethyl Ketone
ketone warheads were examined. Incorporation of a
heteroatom or an electron deficient group at the â′-atom
is one strategy used to enhance the potency of cysteine
protease inhibitors.35-37 Replacing the aldehyde portion
of the warhead of 4 with a methyl thiomethyl ketone
(58) provided a compound with a Ki ) 0.19 µM, which
is 5-fold less active than the corresponding aldehyde 4
(Table 2). However, incorporating the more lipophilic
2-chlorobenzyl thiomethyl ketone warhead (59) resulted
in comparable activity (Ki ) 0.03 µM) relative to 4.
Keto-oxazoles and keto-benzoxazoles have also been
reported as suitable P1 warheads in cysteine protease
inhibitors where the oxazole nitrogen can hydrogen
bond to a histidine side chain in the putative oxyanion
hole of the enzyme. On the basis of work by Vertex in
the area of ICE (caspase-1) inhibitors,34 5-phenyl benz-
oxazole (60) and benzoxazole (61) warheads were in-
corporated into our lead compound with the hope that
we could potentially pick up similar hydrogen-bonding
interactions in the caspase-3 oxyanion hole. Unfortu-
nately, both compounds 60 and 61 exhibited a 10-fold
decrease in activity with Ki values of 0.45 and 0.41 µM,

5012 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Choong et al.
Ta ble 4. Caspase-3 Inhibition by P1 Aspartyl Ketones on a
respectively. The loss of inhibitor potency observed with
these large rigid groups may reflect the more occluded
nature of the caspase-3 active site, which is considerably
more narrow and deep than that of ICE.
Thiophene Scaffold
Lin k er Rep la cem en t w ith Heter ocycles. We ex-
amined the replacement of the pyridyl linker in com-
pound 59 with a variety of heterocycles in the context
of the 2-chlorobenzyl thiomethyl ketone/aldehyde war-
head and the salicylic acid group at P4 (Table 3). The
pyrimidine analogue 62b showed comparable activity
to the corresponding pyridyl analogue 59, Ki ) 0.02 and
0.03 µM, respectively, while pyrazine derivative 63b was
3-fold less active (0.09 µM). The pyridyl linker of 59
could also be successfully replaced with various five-
membered heterocycles. The corresponding 2,5-thiazole
(64b), 2,5-thiophene (66b), and 3,5-isoxazole linkers
(65b) all exhibited excellent in vitro potencies against
caspase-3 with Ki values of 0.03, 0.02, and 0.06 µM,
respectively. Among the various thiophene analogues
66-68a ,b, inhibitory activity seemed to be strongly
dependent on the substitution pattern around the
thiophene ring. Both the 3-aminomethyl-5-carboxy (68b)
and 2-aminomethyl-4-carboxy (67b) analogues were
significantly less potent than the corresponding 2-ami-
nomethyl-5-carboxy analogue 66b. A methyl group
introduced at the 4-position (69b) resulted in compa-
rable activity to 66b , Ki values of 0.04 and 0.02 µM,
respectively. We speculated that substitution at the
4-position with an appropriate binding element could
allow us to pick up additional binding affinity in the S2
subsite of the enzyme. By analogy, introduction of a
methyl group at the 3-position of 66b resulted in a
completely inactive compound (in the context of the
aldehyde warhead; data not shown).
Interestingly, the same substitution pattern on the
furan linker (70a ,b) resulted in compounds with sub-
stantial decreases in potency. This suggests that inhibi-
tor activity in this series can be quite sensitive to the
relative positioning of the groups occupying the S1 and
S4 pockets, given the relatively small differences in
distances and bond angles that distinguish substituents
around furan and the various thiophenes. From the
results seen in Table 3, we selected 66b for further
structure-activity relationship (SAR) studies for rea-
sons of excellent in vitro potency, synthetic accessibility,
and the potential to incorporate and direct substituents
at the 4-position of the thiophene ring for binding into
the S2 subsite of the enzyme.
nitrogen and the side chain of Arg207 in the S3 subsite,
and the same argument can presumably be made for
the pyrimidine analogues 62a ,b. However, in the case
of thiophene analogues (66-68a ,b), no such hydrogen
bond could be formed, so the high affinity binding of
certain members of this series must reflect other
improvements relative to 3. Specifically, the highest
affinity variants in this set may be due to a subtle
repositioning of the key contacts, such as those made
by the S4 salicylic acid group.
Thiomethyl ketones bearing a pyridyl substituent (71)
and (72) were synthesized in order to explore the
possibility of productive polar interactions with residues
such as Glu123 in the S1′ subsite. Both compounds
exhibited excellent in vitro potency but showed no
improvement over 66b. It is interesting to note that the
relatively unactivated phenylpropyl ketone warhead
(73) exhibited only a 10-fold decrease in activity as
compared to the parent compound 66b.
P 1 Wa r h ea d Stu d ies on Th iop h en e An a logu es.
An evaluation of P1 warheads similar to that performed
on the pyridyl linker was conducted on thiophene
analogues 66a ,b (Table 4). In general, SAR of the
thiophene analogues paralleled those of the pyridyl
analogues except in one significant way. There was a
6-fold difference in inhibitory activity observed between
the aldehyde warhead (66a ) and the 2-chlorobenzyl
thiomethyl ketone (66b), Ki ) 0.12 vs 0.02 µM, respec-
tively, which was not observed in the corresponding
pyridyl compounds 4 and 59 or the pyrimidine deriva-
tives 62a ,b (and the other five-membered heterocycles
analogues shown in Table 3). As stated earlier, it was
proposed (and supported by crystallography) that the
gain in potency from 3 to 4 arose from the hydrogen-
bond interaction of the basic amine of the pyridyl
P 4 Mod ifica tion s on Com p ou n d 66b. Compound
66b was selected as a compound for further analogue
studies around the salicylic acid moiety analogous with
that performed on compound 4. Similar truncations of
the salicylic acid group were examined (Table 5), and
the results found are essentially the same as those for
the pyridyl linker. Namely, acidic functionality at the
3-position is critical for good caspase-3 inhibitory activ-

Small Molecule Inhibitors of Caspase-3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5013
Ta ble 7. Crystallographic Data and Refinement Parameters
Ta ble 5. Caspase-3 Inhibition by Substituted P4 Aryl:
Sulfonamides on a Thiophene Scaffold
data
set
space
group
cell constants
R, â, γ (Å)
resolution
(Å)
Rcryst
(%)
Rfree
(%)
3a
C2
123.5, 71.0, 95.9
â ) 136.4°
1.7
15.1
19.2
15.7
18.3
23.9
18.8
4a
C2
124.0, 71.6, 95.8
â ) 136.2°
2.0
66b
I222
71.2, 84.3, 95.5
1.55
a
Crystallization was performed with the irreversible derivatives
of these analogues.
F igu r e 5. Resolution (1.55 Å) X-ray cocrystal stucture of
inhibitor 66b bound within the active site of caspase-3.
Hydrogen-bonding interactions are shown using dashed yellow
lines.
We realized that the sulfonamide linkage connecting the
salicylic acid group to the rest of the molecule was a
potential bottleneck that would hamper our abilities to
fully probe this region of the active site. Therefore, we
decided to look for replacements of the sulfonamide
linkage that would allow us to introduce more readily
available building blocks containing the necessary
functionality to satisfy the hydrogen-bond interactions
observed between the salicylic acid group with the active
site residues in the S4 subsite. Several alternate link-
ages were examined, and the results are shown in Table
6. The reverse sulfonamide (79) would allow the use of
amine building blocks while maintaining the necessary
sulfonamide N-H group for hydrogen bonding. How-
ever, compound 79 was 100-fold less active as compared
to the parent compound 66b. Replacing the sulfonamide
with an amine linkage was also examined. These types
of linkages would allow the introduction of a wide
variety of potential P4 groups by reductive amination
reactions utilizing amine and/or aldehyde building
blocks. The secondary amine (47a ) displayed 40-fold less
activity than the parent compound, but the tertiary
amine (47c) and the corresponding acetamide (47b)
appeared to be well-tolerated with Ki values of 0.09 and
0.14 µM, respectively. Molecular modeling of 47c sug-
gested that the tertiary amine, when protonated, could
make similar hydrogen-bond interactions as that of the
sulfonamide N-H in 66b in the enzyme active site. The
relative importance of this hydrogen-bond interaction
to overall binding affinity is evident in compound 78
where N-methylation results in a 7-fold decrease in
caspase-3 inhibitory activity (Ki ) 0.14 µM). Together,
these results suggest an alternative strategy for intro-
ducing pharmacophores at S4. Through reductive ami-
nation chemistry utilizing amines or aldehydes building
blocks onto the corresponding aldehyde or amine link-
Ta ble 6. Effects on Caspase-3 Inhibition by P3
Sulfonamide-Modified Thiophene Derivatives
ity. Additional analogues in this series were not pursued
based on the lack of success on the pyridyl scaffold.
One obstacle in identifying suitable surrogates for the
salicylic acid group was the small number of com-
mercially available sulfonyl chlorides as well as the lack
of suitable precursors for chlorosulfonylation chemistry.

5014 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Choong et al.
Ta ble 8. Inhibitor Selectivity among Various Caspases
Ki (µM)
Con clu sion s
In this paper, we have described the design, synthesis,
and in vitro activities of a series of potent and selective
nonpeptidic small-molecule inhibitors of caspase-3 that
originated from an initial hit identified from our ex-
tended tethering technology. By using information
obtained from the X-ray crystal structure of the tether-
ing hit in conjunction with molecular modeling studies,
inhibitor 4 was identified as a potent and reversible
inhibitor of caspase-3. With compound 4 as a starting
point for our medicinal chemistry efforts, we were able
to identify additional compounds with improved inhibi-
tory potency and selectivity for caspase-3. Evaluation
of analogues of the salicylic acid group of 4 established
that compounds possessing a carboxylic acid bioisostere
(i.e., tetrazole, acylsulfonamide, or a 5-oxo-1,2,4-oxadia-
zole) exhibited good caspase-3 inhibitory activity. This
result is not surprising since it is known that an aspartic
acid is the preferred residue for the S4 pocket of caspase-
3. In addition, we examined several substituted methyl
ketones as potential aldehyde replacements for the Asp-
CHO warhead. Although this was not looked at exten-
sively, the 2-chlorobenzyl thiomethyl ketone warhead
appeared to give the most potent inhibitory activity
relative to the corresponding aldehyde. The linker
portion of the molecules was extensively examined. It
was shown that various heterocycles such as pyrimidine,
thiazole, thiophene, and isoxazole could be used ef-
fectively as replacements for the pyridine ring of 4.
Interestingly, in the thiophene series of inhibitors
66a ,b -68a ,b , the substitution pattern around the
thiophene was critical for inhibitory activity. Further
optimization of this series resulted in the discovery that
a tertiary amine is an effective replacement for the
sulfonamide group, yielding compound 47c (Ki ) 90 nM).
Finally, a 1.55 Å high resolution X-ray cocrystal struc-
ture of 66b bound within the active site of caspase-3
was determined. Examination of this structure con-
firmed that the key binding interactions of inhibitor 66b
with active site residues are indeed made and are
consistent with the observed SAR.
compd casp-1 casp-2 casp-3 casp-4 casp-5 casp-6 casp-7 casp-8
3
4
47c
59
62b
64b
66a
66b
69b
0.16
3.9
12
1.5
25
8.3
1.9
3.2
2.7
9.0
0.96 0.05
30
11
30
16
21
64
37
0.20
21
92
96
38
22
13
38
12
22
98
11
32
23
24
63
36
1.0
1.4
22
0.47
0.13
0.19
0.15
0.88
4.0
0.09
0.03
0.02
0.03
0.12
0.12 0.09
0.81
0.19 0.09 28
2.3 0.09 19
0.54 0.12
9.7
2.4
0.05
0.33
0.71
0.02 >100
0.04 27
0.13
0.94
ers, a large and diverse set of potential binding elements
can be evaluated.
Cr yst a l St r u ct u r e of 66b Bou n d t o Ca sp a se-3.
The 1.55 Å resolution X-ray cocrystal structure of
inhibitor 66b bound within the active site of caspase-3
was determined (Figure 5, Table 7). As observed in other
inhibitor/caspase-3 structures, the S1 subsite of the
enzyme is occupied by the Asp side chain of the
inhibitor, which is held between the Arg64 (2.8 Å),
Arg207 (2.8 Å), and Gln161 (2.9 Å) residues. As pre-
dicted, the salicylic acid group makes many specific
interactions in the S4 subsite. The 3-carboxyl group
interacts with Trp214 (2.9 Å) and Asn208 (3.1 Å), and
the 4-hydroxyl interacts with the main chain N-H of
Phe250. The sulfonamide N-H and one of the oxygens
closely interact with the main chain oxygen of Arg207
(2.9 Å) and the main chain N-H of Ser209 (3.1 Å),
respectively. Finally, the main chain N-H of the P1
aspartyl group forms a hydrogen bond with the carbonyl
of Ser205 (3.1 Å). The S1′ subsite is occupied by the
2-chlorobenzyl ring of the thiomethyl ketone warhead,
which does not appear to be making any specific
interactions with the enzyme. This high resolution
structure reveals the remarkable manner in which this
caspase inhibitor, discovered through tethering technol-
ogy, is able to effectively mimic seven of the eight
hydrogen-bonding interactions seen in peptide inhibi-
tors, such as AcDVAD-CHO.39
Selectivity Stu d ies. The nine most active com-
pounds were screened for selectivity studies against
caspase-1-8. In general, these compounds are very poor
inhibitors of caspase-1, -2, -4, and -5, indicating that our
compounds are highly specific for inhibiting the apop-
totic caspases (3 and 6-8) and not the inflammatory
caspases (1 and 4). As shown in Table 8, compound 3,
which possesses the simple phenyl linker, has only
moderate selectivity between the caspases; however,
inhibitors that incorporate a heterocyclic linker show
200-3000-fold selectivity for caspase-3 over caspase-1
(and others relative to compound 3). In only two cases
were compounds relatively active against caspase-6
(compounds 59 and 62b). Even though caspase-3 and
caspase-7 have almost identical cleavage specificities,9
we surprisingly found that some compounds showed
selectivity for caspase-3, e.g., compounds 59 and 62b
show ∼3-5-fold selectivity. These compounds therefore
represent some of the most specific nonpeptidic com-
pounds for caspase-3 reported to date, with the only
other reported compounds that showed specificity being
the isatin series reported by SmithKline Beecham (∼4-
fold selectivity for caspase-3 vs caspase-7).39
In summary, the application of extended tethering to
caspase-3 yielded a novel, weak binding fragment (a
salicylic acid), which exhibited appreciable binding
interactions with the S4 pocket of the active site.
Exploitation of this weak binding fragment eventually
led to the discovery of a series of potent, selective, and
reversible nonpeptidic inhibitors of caspase-3.
Exp er im en ta l Section
Gen er a l Meth od s. Unless otherwise noted, all reactions
were conducted under a nitrogen atmosphere. All commercially
available starting materials and solvents were reagent grade
or better and used without further purification. Solutions
containing products were dried over anhydrous magnesium
sulfate (MgSO4) or sodium sulfate (Na2SO4) followed by
vacuum filtration. Flash column chromatography was carried
out using Merck Kieselgel 60 silica gel (230-400 mesh). Solid-
phase reactions were conducted on aminomethylated polysty-
rene HL (100-200 mesh) from Novabiochem in Alltech 8 or
25 mL Extract-Clean reservoirs fitted with the corresponding
frits and caps with agitation supplied by an orbital shaker
table and a Supelco Vacuum Manifold used for resin washing.
Preparative high-performance liquid chromatography (HPLC)
purification was carried out on a Gilson HPLC fitted with a
Waters Nova-Pak C-18 (25 mm × 100 mm) column eluting at

Small Molecule Inhibitors of Caspase-3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5015
25 mL/min with a gradient of 10-100% acetonitrile in water
(0.1% TFA) over 10 min and holding at 100% for 3 min.
Fractions were pooled and lyophilized to provide final products
as the free acid or TFA salt as indicated. 1H NMR spectroscopy
was determined on a 400 MHz Bruker spectrometer with
chemical shifts reported in units of parts per million (ppm).
High-resolution mass spectra (HRMS) were determined on a
Applied Biosystems QStar Pulsar-i. Elemental analyses were
carried out by Robertson Microlit Laboratories Inc., Madison,
NJ .
with water and brine, dried, and concentrated in vacuo to give
a residue that was dissolved in xylene (50 mL). The solution
was heated under reflux for 3 h, concentrated under reduced
pressure, and partitioned between water and ethyl acetate.
The organic layer was separated, washed with water and
brine, and dried. Concentration in vacuo followed by purifica-
tion of the residue by flash column chromatography (10% ethyl
acetate to 50% ethyl acetate in hexanes) afforded the 5-oxo-
1,2,4-oxadiazole derivative as an off-white powder (1.95 g,
1
70%). H NMR (DMSO-d6): δ 12.90 (s, 1H), 9.63 (s, 1H), 8.05
(s, 1H), 7.54 (d, 1H), 7.43 (t, 1H), 7.35 (d, 1H), 1.47 (s, 9H).
This material (831 mg, 3.0 mmol) was dissolved in anhydrous
4 N HCl in dioxane (6 mL) and stirred for 3 h at room
temperature. The solution was concentrated to dryness and
of sufficient purity for use in the next step without further
purification. This intermediate was subjected to previously
described chlorosulfonylation conditions to provide compound
12 as a yellow solid (159 mg, 61%). 1H NMR (DMSO-d6): δ
13.04 (s, 1H), 8.10 (s, 1H), 7.85 (d, J ) 7.5 Hz, 1H), 7.75 (d, J
) 7.5 Hz, 1H), 7.54 (t, J ) 7.7 Hz, 1H).
Gen er a l P r oced u r e for Ch lor osu lfon yla tion of Su b-
stitu ted An ilin es 5-9. An appropriately substituted aniline
(5.0 mmol) was dissolved in concentrated HCl (5 mL) and
water (2 mL) followed by the addition of glacial acetic acid (5
mL) to give a yellow solution, which was cooled to -10 °C. A
solution of NaNO2 (6.0 mmol) in water (3 mL) was added
dropwise while maintaining the temperature below -5 °C. The
resulting mixture was stirred at -5 °C for 15 min and added
to a cooled (5 °C) solution of glacial acetic acid (10 mL)
containing CuCl (1.25 mmol) that was previously saturated
with SO2 gas for 45 min. After vigorous gas evolution, the
resulting green mixture was stirred for 1-2 h while allowing
the reaction to warm to room temperature. Excess SO2 and
acetic acid were removed under reduced pressure, and the
desired sulfonyl chloride was isolated by either precipitation
from water or extraction with ethyl ether and washing with 1
N HCl, water, and brine and drying. Using this method,
compounds 5-9 were obtained and of sufficient purity for use
in the next step without further purification.
6-Br om om eth yl-n icotin ic Acid Meth yl Ester (14). To
a solution of 13 (5.0 g, 33 mmol) in benzene (110 mL) were
added N-bromosuccinimide (5.3 g, 30 mmol), benzoyl peroxide
(0.801 g, 3.31 mmol), and acetic acid (2 mL). After it was
heated at reflux for 9 h, the solution was diluted with ethyl
acetate and washed with 1 N HCl. The organic layer was
separated, dried, and concentrated in vacuo to give a dark
residue. Purification by flash column chromatography (10%
1
ethyl acetate in hexanes) afforded 14 (2.88 g, 38%). H NMR
(CDCl3): δ 9.17 (s, 1H), 8.31 (dd, J ) 8.1, 2.1 Hz, 1H), 7.53 (d,
J ) 8.1 Hz, 1H), 4.60 (s, 2H), 3.96 (s, 3H). EI-MS m/z: 232.1
(M + H)+.
3-Meth an esu lfon ylam in o-ben zen esu lfon yl Ch lor ide (5).
Compound 5 was prepared from N-(3-amino-phenyl)methane-
sulfonamide as an oil (120 mg, 15%).
4-Meth an esu lfon ylam in o-ben zen esu lfon yl Ch lor ide (6).
Compound 6 was prepared from N-(4-amino-phenyl)methane-
sulfonamide as an oil (200 mg, 25%).
6-Am in om eth yl-n icotin ic Acid Meth yl Ester (15). A
solution of 14 (2.86 g, 12.8 mmol) and sodium azide (1.09 g,
16.7 mmol) in DMF (64 mL) was heated at 70 °C for 30 min.
The reaction mixture was diluted with ethyl acetate and
washed with saturated NaHCO3. The organic layer was
separated, dried, and concentrated in vacuo to afford the azide
in 69% yield. A solution of this material (1.66 g, 8.60 mmol)
and 10% palladium on carbon (200 mg) in absolute ethanol
(20 mL) was stirred under a 20 psi hydrogen atmosphere for
20 min. The reaction mixture was filtered through a pad of
Celite and washed with CH2Cl2. The filtrate was concentrated
in vacuo to afford 15 as an orange oil of sufficient purity for
use in the next step without further purification. EI-MS m/z:
167.1 (M + H)+.
3-Met h a n esu lfon yla m in oca r b on yl-b en zen esu lfon yl
Ch lor id e (7). To a mixture of 3-tert-butoxycarbonylamino-
benzoic acid (1.19 g, 5.00 mmol), methanesulfonamide (0.62
g, 6.50 mmol), and N,N-(dimethylamino)pyridine (DMAP) (0.79
g, 6.50 mmol) in CH2Cl2 (17 mL) was added EDC (1.25 g, 6.50
mmol). The reaction mixture was stirred at room temperature
for 24 h, concentrated in vacuo, and purified by flash column
chromatography (40% ethyl acetate in hexanes with 1% acetic
acid) to afford the acylsulfonamide as a white solid (1.5 g, 95%).
1H NMR (CDCl3): δ 7.94 (s, 1H), 7.54 (bd, 2H), 7.40 (m, 1H),
6.80 (bs, 1H), 3.42 (s, 3H), 1.53 (s, 9H). This material was
treated with 4 N HCl in dioxane for 2 h at room temperature,
concentrated in vacuo, and subjected to previously described
chlorosulfonylation conditions to provide 7 as a white solid (376
mg, 63%). 1H NMR (CDCl3): δ 8.51 (s, 1H), 8.22 (d, J ) 7.4
Hz, 1H), 8.16 (d, J ) 7.5 Hz, 1H), 7.69 (t, J ) 7.7 Hz, 1H),
3.30 (s, 3H).
6-[(9H -F lu or en -9-ylm et h oxyca r b on yla m in o)m et h yl]-
n icotin ic Acid Hyd r och lor id e (16). A solution of 15 (770
mg, 4.63 mmol) in dioxane (15 mL) and 1 M LiOH (12 mL)
was stirred at room temperature for 5 h, acidified with 1 N
HCl, and concentrated to dryness to afford the amino acid as
a yellow solid. The crude solid was dissolved in dioxane (20
mL) and water (20 mL) followed by the addition of NaHCO3
(7.80 g, 46.3 mmol) and Fmoc-OSu (1.72 g, 5.10 mmol). After
it was stirred at room temperature for 22 h, the reaction was
concentrated to dryness, diluted with water, and acidified to
pH 1 with 2 N HCl to give the desired product as an off-white
solid, which was collected on a sintered glass funnel, washed
with water, and dried to provide 16 as an off-white powder
3-Acetylsu lfa m oyl-ben zen esu lfon yl Ch lor id e (8). Com-
pound 8 was prepared from N-acetyl-3-amino-benzenesulfona-
1
mide as a yellow powder (458 mg, 51%). H NMR (DMSO-d6):
δ 12.12 (s, 1H), 8.09 (s, 1H), 7.85 (m, 2H), 7.58 (t, J ) 7.8 Hz,
1H), 1.92 (s, 3H). EI-MS m/z: 320.0 (M + Na)+.
3-(1H-Tetr a zol-5-yl)ben zen esu lfon yl Ch lor id e (9). Com-
pound 9 was prepared from 3-(1H-tetrazol-5-yl)aniline as a
1
1
(1.67 g, 88%). H NMR (CD3OD): δ 7.79 (d, J ) 7.4 Hz, 2H),
pale yellow solid (832 mg, 68%). H NMR (DMSO): δ 10.5 (s,
7.65 (d, J ) 7.7 Hz, 2H), 7.34-7.57 (m, 3H), 7.30 (t, J ) 7.4
Hz, 2H), 7.15 (d, J ) 7.7 Hz, 1H), 4.40 (d, J ) 6.6 Hz, 2H),
4.31 (s, 2H), 4.20-4.22 (m, 1H). EI-MS m/z: 375.0 (M + H)+.
1H), 8.32 (s, 1H), 7.99 (d, J ) 7.7 Hz, 1H), 7.78 (d, J ) 7.8 Hz,
1H), 7.55 (t, J ) 7.7 Hz, 1H). EI-MS m/z: 245.0 (M + H)+.
3-(5-Oxo-4, 5-d ih yd r o[1,2,4]oxa d ia zol-3-yl)b en zen e-
su lfon yl Ch lor id e (12). To a solution of 10 (6.54 g, 30.0 mmol)
in ethanol (60 mL) was added NH2OH (3.6 mL, 60 mmol of a
50% w/w solution in water). The reaction was refluxed at 80
°C for 3 h and concentrated to dryness to afford the N-hydroxy
amidine as a yellow solid (7.5 g, 99%) of sufficient purity for
use in the next step without further purification. 2-Ethylhexyl
chloroformate (2.9 mL, 15 mmol) was added dropwise to a
mixture of this material (2.51 g, 10.0 mmol) and pyridine (1.21
mL, 15.0 mmol) in THF (20 mL) at 0 °C. After it was stirred
for 30 min, the reaction mixture was diluted with water and
extracted with ethyl acetate. The organic layer was washed
5-F or m yl-th iop h en e-2-ca r boxylic Acid ter t-Bu tyl Ester
(18). To a solution of 17 (18.0 g, 94.3 mmol) and 2,3-
dimethylbutene (22 mL, 189 mmol) in tert-butyl alcohol (470
mL) was added a solution of NaClO2 (10.6 g, 118 mmol) and
NaH2PO4 (14.1 g, 118 mmol) in water (94 mL). After it was
stirred at room temperature for 12 h, the organic solvent was
removed under reduced pressure and the remaining slurry was
basified with 1 N NaOH (200 mL). The aqueous layer was
washed with several portions of hexanes followed by acidifica-
tion with 1 N HCl and extraction of the desired compound into
ether. The organic layer was dried and concentrated in vacuo

5016 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Choong et al.
to afford the desired product of sufficient purity for use in the
next step without further purification. ES-MS m/z: 208.9 (M
+ H)+. To a solution of this material in CH2Cl2 (316 mL) was
added tert-butyl alcohol (116 mL, 1.30 mol), EDC (24.3 g, 12.7
mmol), and DMAP (7.8 g, 63.3 mmol). After it was stirred for
3 h at room temperature, the solution was diluted with CH2-
Cl2 and washed with 1 N HCl. The organic layer was dried,
concentrated in vacuo, and purified by flash column chroma-
tography (30% ethyl acetate in hexanes) to afford compound
18 (12.5 g, 50% for two steps). EI-MS m/z: 206.9 (M - t-Bu)+.
5-F or m yl-th iop h en e-2-ca r boxylic Acid ter t-Bu tyl Ester
(19). To a solution of 18 (7.5 g, 28.5 mmol) in ether (60 mL) at
-78 °C was added n-butyllithium (32.1 mL, 25.7 mmol, 0.8 M
in hexanes) dropwise. After it was stirred for 1 h at -78 °C, a
solution of 1-formylpiperidine (4.7 mL, 42.8 mmol) in ether
(10 mL) was added dropwise. After it was stirred for an
additional 30 min at -78 °C, the reaction was quenched with
1 N HCl and allowed to warm to room temperature. The
solution was diluted with ether, washed with 1 N HCl, dried,
concentrated in vacuo, and purified by flash column chroma-
tography (30% ethyl acetate in hexanes) to afford compound
19 (3.0 g, 50%). EI-MS m/z: 157.1 (M + H)+.
5-Am in om et h yl-t h iop h en e-2-ca r b oxylic Acid ter t-
Bu tyl Ester (20). To a solution of 19 (3.0 g, 14.1 mmol) in
MeOH (15 mL) was added aqueous NH2OH (1.3 mL, 2.1 mmol,
16.3 M in water). After it was stirred for 1 h at room
temperature, the solvent was removed under reduced pressure
to provide the crude oxime of sufficient purity for use in the
next step without further purification. ES-MS m/z: 172.1 (M
+ H)+. To a solution of this material in acetic acid (28 mL) at
0 °C was added zinc dust (3.6 g, 56.4 mmol). The heterogeneous
mixture was warmed to room temperature and stirred for an
additional 3 h. The reaction was filtered through a pad of
Celite and concentrated in vacuo to provide the crude amine
of sufficient purity for use in the next step without further
purification. EI-MS m/z: 141.1 (M - t-Bu - NH2)+.
5-[(9H -F lu or en -9-ylm et h oxyca r b on yla m in o)m et h yl]-
th iop h en e-2-ca r boxylic Acid (21). To a solution of amine
20 (14.1 mmol) in dioxane (30 mL) and water (30 mL) was
added NaHCO3 (5.9 g, 70.5 mmol) and Fmoc-OSu (5.2 g, 15.5
mmol). After it was stirred at room temperature for 3 h, the
heterogeneous mixture was diluted with ether and washed
with 1 N HCl. The organic layer was dried, concentrated in
vacuo, and purified by flash column chromatography (30%
ethyl acetate in hexanes) to provide the Fmoc-protected tert-
butyl ester (4.62 g, 75%). EI-MS m/z: 458.0 (M + Na)+. A
solution of this material in TFA/CH2Cl2/water (1:4:0.1, 30 mL)
was stirred at room temperature for 2 h and evaporated to
dryness to afford 21, which was of sufficient purity for use in
the next step without further purification. 1H NMR (CD3OD):
δ 7.78 (d, J ) 7.4 Hz, 2H), 7.60-7.65 (m, 3H), 7.38 (t, J ) 7.3
Hz, 2H), 7.29 (t, J ) 7.3 Hz, 2H), 6.95 (s, 1H), 4.45 (s, 2H),
4.40 (d, J ) 6.8 Hz, 2H), 4.21 (t, J ) 6.7 Hz, 1H). EI-MS m/z:
402.0 (M + Na)+.
mmol). After it was stirred for 30 min at room temperature,
the solution was diluted with CH2Cl2, washed with 1 N HCl,
dried, and concentrated in vacuo. Purification by flash column
chromatography (100% hexanes to 10% ethyl acetate in
hexanes) afforded 24 (10.6 g, 92% for two steps).
5-(ter t-Bu tyl-d im eth yl-sila n yloxym eth yl)th iop h en e-3-
ca r boxylic Acid Eth yl Ester (25). To a solution of 24 (10.6
g, 34.3 mmol) in THF (60 mL) at -78 °C was added n-
butyllithium (23.6 mL, 37.8 mmol, 1.6 M in hexanes) dropwise.
After it was stirred at -78 °C for 1 h, the reaction solution
was added dropwise to a solution of ethyl chloroformate (4.3
mL, 44.7 mmol) in THF (80 mL) at -78 °C via cannula. After
it was stirred for 1 h, the reaction was quenched with 1 N
HCl and warmed to room temperature. The organic layer was
separated, washed with 1 N HCl, and dried. Concentration in
vacuo followed by purification by flash column chromatography
(10% ethyl acetate in hexanes) afforded 25 (10.1 g, 97%).
5-Br om om eth yl-th iop h en e-3-ca r boxylic Acid Eth yl Es-
ter (26). To a solution of 25 (10.1 g, 33.6 mmol) in THF (70
mL) was added acetic acid (12 mL) and tetrabutylammonium
fluoride (TBAF) (36.9 mL, 36.9 mmol of a 1.0 M solution in
THF). After it was stirred for 12 h, the solution was diluted
with ether, washed with saturated NaHCO3, and dried.
Concentration in vacuo followed by flash column chromatog-
raphy (30% ethyl acetate in hexanes) afforded the desired
alcohol. EI-MS m/z: 187.0 (M + H)+. To a solution of the
alcohol (3.0 g, 16.1 mmol) in THF (80 mL) was added CBr4
(5.9 g, 17.7 mmol) and triphenylphosphine (4.6 g, 17.7 mmol).
After it was stirred for 1 h, the suspension was filtered,
concentrated in vacuo, and purified by flash column chroma-
tography (20% ethyl acetate in hexanes) to provide bromide
26 (4.0 g, 100%). EI-MS m/z: 250.9 (M + H)+.
5-Am in om eth yl-th iop h en e-3-ca r boxylic Acid Eth yl Es-
ter (27). A solution of 26 (4.0 g, 16.1 mmol) and sodium azide
(1.2 g, 17.7 mmol) in DMF (30 mL) was stirred at 50 °C for 30
min. The reaction mixture was diluted with ether, washed with
water, dried, and concentrated in vacuo to provide 1.9 g (56%)
of the azide, which was of sufficient purity for use in the next
step without further purification. EI-MS m/z: 212.9 (M + H)+.
A solution of this material (1.9 g, 9.0 mmol) and 10% palladium
on carbon (100 mg) in absolute ethanol (30 mL) was hydro-
genated at 30 psi for 2 h. The suspension was filtered through
a pad of Celite and concentrated in vacuo to provide amine 27
(900 mg, 52%), which was of sufficient purity for use in the
next step without further purification. EI-MS m/z: 169.1 (M
- NH2)+.
5-[(9H -F lu or en -9-ylm et h oxyca r b on yla m in o)m et h yl]-
th iop h en e-3-ca r boxylic Acid (28). To a solution of 27 (0.9
g, 4.9 mmol) in dioxane (10 mL) was added 1 M LiOH (10 mL).
The reaction was stirred for 1 h at room temperature,
neutralized with 1 N HCl, and concentrated in vacuo to provide
the amino acid of sufficient purity for use in the next step
without further purification. To a solution of this material in
dioxane (5 mL) and water (5 mL) was added NaHCO3 (2.1 g,
24.5 mmol) and Fmoc-OSu (1.8 g, 5.4 mmol). After it was
stirred for 3 h at room temperature, the solution was diluted
with ether, washed with 1 N HCl, dried, and concentrated in
vacuo. Purification of the residue by flash column chromatog-
raphy (5% methanol in ethyl acetate) afforded 28. 1H NMR
(CDCl3): δ 8.04 (s, 1H), 7.78 (d, J ) 7.4 Hz, 3H), 7.63 (d, J )
7.4 Hz, 2H), 7.28-7.39 (m, 4H), 4.43 (d, J ) 4.8 Hz, 2H), 4.36
(J ) 6.9 Hz, 2H), 4.20 (t, J ) 6.8 Hz, 1H). EI-MS m/z: 402.3
(M)+.
5-[(9H -F lu or en -9-ylm et h oxyca r b on yla m in o)m et h yl]-
th iop h en e-2-ca r boxylic Acid (22).40 To a solution of 21
(0.500 g, 1.3 mmol) in TFA/CHCl3 (1:1, 15 mL) was added 37%
formaldehyde (1.2 mL, 14 mmol) in water. After it was stirred
at room temperature for 30 min, triethylsilane (2.9 mL, 18
mmol) was added and the solution was stirred for an additional
30 min. The solvent was concentrated in vacuo, and the crude
product was purified by flash column chromatography (100%
ethyl acetate) to afford 22 0.510 g, 100%). EI-MS m/z: 416.2
(M + Na)+.
(4-Br om o-th iop h en -2-ylm eth oxy)-ter t-bu tyl-d im eth yl-
sila n e (24). To a solution of 23 (7.1 g, 37.2 mmol) in THF (80
mL) was added NaBH4 (1.5 g, 40.9 mmol) in portions. After it
was stirred for 30 min, saturated NaHCO3 was slowly added
to quench the reaction. The reaction mixture was diluted with
ether, washed with saturated NaHCO3, dried, and concen-
trated in vacuo to provide the alcohol, which was of sufficient
purity for use in the next step without further purification.
To a solution of this material (37.1 mmol) in CH2Cl2 (70 mL)
was added TBSCl (6.1 g, 40.8 mmol) and imidazole (2.8 g, 40.8
5-(2-Ch lor o-b en zylsu lfa n yl)-3-(9H -flu or en -9-ylm et h -
oxyca r b on yla m in o)-4-oxo-p en t a n oic Acid ter t-Bu t yl
Ester (30). To a solution of Fmoc-Asp(Ot-Bu)-CH2Br20 (20.3
g, 48.4 mmol) in THF (150 mL) was added 2-chlorobenzene-
methanethiol (6.9 mL, 53.2 mmol) and diisopropylethylamine
(9.3 mL, 53.2 mmol). After 1 h, the solution was diluted with
ethyl acetate and washed with saturated NaHCO3. The organic
layer was dried and concentrated in vacuo to provide 29 (24.1
g, 100%), which was used without further purification. 1H

Small Molecule Inhibitors of Caspase-3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5017
NMR (CDCl3): δ 7.75 (d, J ) 7.4 Hz, 1H), 7.57 (d, J ) 7.3 Hz,
1H), 7.28-7.41 (m, 8H), 3.78-3.83 (m, 5H). ES-MS m/z: 498.2
(M + H)+.
Ester (39). To a solution of 37 (18.76 g) in acetonitrile (41
mL) was added NaN3 (9.39 g, 144 mmol) followed by stirring
at room temperature for 3 days. The reaction was diluted with
CH2Cl2, washed with water, dried, and concentrated in vacuo
to provide the azide (14.1 g, 98%), which was used without
further purification. ES-MS m/z: 198 (M + H)+. To a solution
of the azide (7.48 g, 38.0 mmol) in acetic acid (75 mL) was
added zinc dust (15.1 g, 232 mmol) portionwise. After it was
stirred for 2 h, the reaction was filtered through a pad of Celite
and washed with acetic acid. The combined filtrates were
concentrated in vacuo to provide the crude amine (14.4 g). To
a solution of the amine (14.4 g) in CH2Cl2 (70 mL) was added
NMM (12.5 mL, 124 mmol) followed by 5-chlorosulfonyl-2-
hydroxybenzoic acid (8.66 g, 36.7 mmol) and stirred for 2 h.
The solution was diluted with CH2Cl2, washed with 1 N HCl,
dried, and concentrated in vacuo to give the crude 38, which
was used directly in the next step. A solution of this material
was suspended in toluene (60 mL) and heated to 90 °C. DMF
di-tert-butyl acetal28 (35 mL, 146.0 mmol) was dropwise added
to the hot solution. After it was stirred at 90 °C for 1.5 h, the
solution was diluted with CH2Cl2, washed with 1 N HCl, dried,
and concentrated in vacuo. Flash chromatography over silica
gel (25% ethyl acetate in hexanes) provided 39 (4.19 g, 26%
after three steps). 1H NMR indicated that the product com-
prised a 1:1 mixture of the tert-butyl ester and the O-tert-butyl
phenol. ES-MS m/z: 450 (M + Na)+.
5-[(3-ter t-Bu t oxyca r b on yl-4-h yd r oxy-b en zen esu lfon -
yla m in o)m eth yl]th iop h en e-2-ca r boxylic Acid (40). To a
solution of 39 (4.19 g, 9.84 mmol) in dioxane (40 mL) was added
1 M LiOH (40 mL). After it was stirred at room temperature
for 45 min, the solution was diluted with ether, washed with
1 N HCl, dried, and concentrated in vacuo to give 40 (4.73 g,
117%) that still contained some residual dioxane. 1H NMR
(CDCl3): δ 11.63 (s, 0.5H), 8.26 (d, J ) 2.4 Hz, 0.5H), 8.04 (d,
J ) 2.4 Hz, 0.5H), 7.88 (dd, J ) 8.7, 2.4 Hz, 0.5H), 7.80 (dd, J
) 8.8, 2.5 Hz, 0.5H), 7.66 (d, J ) 3.8 Hz, 0.5H), 7.64 (d, J )
3.8 Hz, 0.5H), 7.17 (d, J ) 8.8 Hz, 0.5H), 7.06 (d, J ) 8.8 Hz,
0.5H), 6.92 (d, J ) 4.7 Hz, 0.5H), 6.92 (d, J ) 4.2 Hz, 0.5H),
4.95 (t, J ) 6.4 Hz, 0.5H), 4.92 (t, J ) 6.3 Hz, 0.5H), 4.39 (d,
3-(9H -F lu or en -9-ylm et h oxyca r b on yla m in o)-4-oxo-5-
(p yr id in -3-ylm eth ylsu lfa n yl)p en ta n oic Acid ter t-Bu tyl
Ester (33a ). 3-Picolyl mercaptan (32a ) was prepared from
3-picolyl chloride hydrochloride (31a ) by literature methods.30
The corresponding thiomethyl ketone was prepared using the
same method as 30 except for using 3-picolyl mercaptan as a
reagent instead of 2-chlorobenzenemethanethiol. ES-MS m/z:
533 (M + H)+.
3-(9H -F lu or en -9-ylm et h oxyca r b on yla m in o)-4-oxo-5-
(p yr id in -4-ylm eth ylsu lfa n yl)p en ta n oic Acid ter t-Bu tyl
Ester (33b). 4-Picolyl mercaptan (32b) was prepared from
4-picolyl chloride hydrochloride (31b) by literature methods.30
The corresponding thiomethyl ketone was prepared using the
same method as 30 except for using 4-picolyl mercaptan as a
reagent instead of 2-chlorobenzenemethanethiol. ES-MS m/z:
533 (M + H)+.
3-(9H -F lu or en -9-ylm et h oxyca r b on yla m in o)-4-oxo-7-
p h en yl-h ep ta n oic Acid ter t-Bu tyl Ester (35). To a solution
of 34 (1.8 mL, 14.5 mmol) in anhydrous THF (10 mL) at -78
°C was added n-butyllithium (9.08 mL, 14.5 mmol, 1.6 M in
hexanes) dropwise over a 10 min period. After it was stirred
for 15 min, a solution of N-Fmoc-Asp(Ot-Bu)-CHO (2.61 g, 6.6
mmol) in anhydrous THF (20 mL) was added dropwise over a
15 min period. The solution was stirred at -78 °C for an
additional 40 min, quenched with water, and warmed to room
temperature. The reaction mixture was diluted with ether,
washed with 1 N HCl, dried, and concentrated in vacuo.
Purification by flash column chromatography (30% ethyl
acetate to 40% ethyl acetate in hexanes) provided the alcohol
(890 mg, 27%). A solution of this material (890 mg, 3.91 mmol)
and 10% palladium on carbon (125 mg) in absolute ethanol
(15 mL) was hydrogenated at atmospheric pressure for 1 h.
The suspension was filtered through a pad of Celite and
washed with ethanol. The combined filtrates were concen-
trated in vacuo to provide the alcohol in quantitative yield.
To a solution of this material (700 mg, 1.36 mmol) in CH2Cl2
(7 mL) was added Dess-Martin periodinane (865 mg, 2.04
mmol) in one portion at room temperature. After it was stirred
for 30 min, the solid was filtered and washed with CH2Cl2.
The combined filtrates were concentrated in vacuo and purified
by flash column chromatography (25% ethyl acetate in hex-
anes) to afford 35 (325 mg, 47%). 1H NMR (CDCl3): δ 7.77 (d,
J ) 7.6 Hz, 2H), 7.59 (d, J ) 7.1 Hz, 2H), 7.41 (t, J ) 7.5 Hz,
2H), 7.31 (t, J ) 7.1 Hz, 2H), 7.26 (m, 2H), 7.17 (t, J ) 7.2 Hz,
3H), 5.87 (d, J ) 7.8 Hz, 1H), 4.43 (m, 2H), 4.22 (t, J ) 7.0
Hz, 1H), 2.88 (m, 1H), 2.70 (dd, J ) 17.1, 4.7 Hz, 1H), 2.58
(m, 4H), 1.93 (m, 2H), 1.42 (s, 9H).
J
) 6.3 Hz, 0.5H), 3.70 (s, 3H), 1.64 (s, 4.5H), 1.49 (s,
4.5H).
5-[(5-{1-Ca r b oxym e t h yl-2-[5-(2,6-d ich lor o-p h e n yl)-
oxa zol-2-yl]-2-oxo-eth ylca r ba m oyl}th ioph en -2-ylm eth yl)-
su lfa m oyl]-2-h yd r oxy-b en zoic Acid (41b ). The phenyl-
oxazole amino alcohol 41a was prepared according to literature
procedure.34 To a solution of 41a (0.138 mmol) and 40 (54.9
mg, 0.142 mmol) in DMF (1.4 mL) was added 1-hydroxyben-
zotriazole (HOBt) (20.3 mg, 0.150 mmol) and EDC (26.9 mg,
0.140 mmol). After it was stirred at room temperature for 1
h, the solution was diluted with ether and washed with water.
The organic layer was separated, dried, concentrated in vacuo,
and purified by flash column chromatography (50% ethyl
acetate in hexanes) to afford the coupled product (80 mg, 74%),
which was dissolved in CH2Cl2 (1.5 mL) and treated with
Dess-Martin periodinane (49.4 mg, 0.116 mmol). The reaction
was stirred for 2 h, washed with 1 N HCl, dried, and
concentrated in vacuo to provide the ketone (106 mg), which
was stirred at room temperature in TFA/CH2Cl2 (1:1, 2 mL)
for 1.5 h. Volatiles were removed under reduced pressure, and
the residue purified by preparative HPLC to provide 41b (30
mg, 30% for two steps). EI-MS m/z: 668.0 (M + H)+. HRMS
(TOF): exact mass calcd for C26H20N3O10S2Cl2 (M + H)+,
667.9961; found, 667.9995.
5-({5-[1-Ca r boxym eth yl-2-(7-m eth yl-ben zoxa zol-2-yl)-
2-oxo-eth ylca r ba m oyl]th iop h en -2-ylm eth yl}su lfa m oyl)-
2-h yd r oxy-ben zoic Acid (42b). The benzoxazole amino
alcohol 42a was prepared according to literature procedure.34
Compound 42b was prepared from 42a and 40 in the same
manner as described for 41b. 1H NMR (acetone-d6): δ 8.35 (d,
J ) 1.8 Hz, 1H), 8.06 (d, J ) 7.9 Hz, 1H), 7.96 (d, J ) 10.8 Hz,
1H), 7.88 (d, J ) 8.3 Hz, 1H), 7.20-7.60 (m, 3H), 7.20 (d, J )
8.1 Hz, 1H), 7.11 (d, J ) 9.0 Hz, 1H), 6.94 (d, J ) 4.5 Hz, 1H),
5.86 (m, 1H), 4.38 (m, 3H), 4.05 (s, 3H), 3.30 (dd, J ) 17.6, 6.0
Hz, 1H), 3.14 (dd, J ) 17.6, 7.4 Hz, 1H). ES-MS m/z: 604 (M
5-Br om om eth yl-th iop h en e-2-ca r boxylic Acid Meth yl
Ester (37). To a solution of 5-methylthiophene-2-carboxylic
acid 36 (20.0 g, 141 mmol) in benzene (120 mL) and methanol
(14 mL) at 0 °C was dropwise added 2.0 M trimethylsilyldia-
zomethane in hexanes (80 mL, 160 mmol). The reaction was
warmed to room temperature, and the solvent was concen-
trated in vacuo. Distillation under reduced pressure (BP0.5
)
88-93 °C) provided the methyl ester as a colorless oil (17.95
1
g, 82%). H NMR (CDCl3): δ 7.61 (d, J ) 3.7 Hz, 1H), 6.76 (d,
J ) 3.6 Hz, 1H), 3.85 (s, 3H), 2.52 (s, 3H). To a solution of the
methyl ester (11.4 g, 72.9 mmol) in CCl4 (200 mL) was added
NBS (14.3 g, 80.3 mmol) and 2,2′-azobisisobutyronitrile (AIBN)
(91 mg, 0.55 mmol). The solution was heated to reflux for 2 h,
at which point 1H NMR of a filtered aliquot indicated a 1:1
ratio of starting material to product. Another portion of AIBN
(81 mg, 0.49 mmol) was added, and the solution was refluxed
for another 2 h. The reaction was then cooled to room
temperature, filtered, and concentrated in vacuo to provide
37 as an orange oil (18.8 g, 110%) that contained =10% of the
1
5-dibromomethyl analogue. H NMR (CDCl3): δ 7.63 (d, J )
3.8 Hz, 1H), 7.09 (d, J ) 3.8 Hz, 1H), 4.67 (s, 2H), 3.88 (s,
3H).
5-[(3-ter t-Bu t oxyca r b on yl-4-h yd r oxy-b en zen esu lfon -
yla m in o)m et h yl]t h iop h en e-2-ca r b oxylic Acid Met h yl

5018 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Choong et al.
+ H)+. HRMS (TOF): exact mass calcd for C25H22N3O11S2 (M
+ H)+, 604.0690; found, 604.0719.
r id in -2-ylm eth yl]su lfa m oyl}-2-h yd r oxy-ben zoic Acid Tr i-
flu or oa ceta te (4). Compound 4 was prepared from resin 44e
(5.4 mg, 6%). H NMR (CD3OD): δ 8.94 (s, 1H), 8.45 (d, J )
1
N -{1-[5-(2,6-Dich lor o-p h e n yl)oxa zole -2-ca r b on yl]-
p r op yl}-6-[(4-h yd r oxy-3-m eth yl-ben zen esu lfon yla m in o)-
m eth yl]n icotin a m id e (60). The advanced nicotinate inter-
mediate was prepared in the same manner as 40 except using
6-methylnicotinic acid as a reagent instead of 36. Compound
60 was prepared using this advanced nicotinate intermediate
and 41a 34 (21 mg, 14%) in the same manner as described for
41b. 1H NMR (acetone-d6): δ 8.94 (s, 1H), 8.48 (d, J ) 7.4 Hz,
1H), 8.36 (d, J ) 2.4 Hz, 1H), 8.20 (d, J ) 8.0 Hz, 1H), 8.00
(dd, J ) 9.0, 2.5 Hz, 1H), 7.55-7.80 (m, 5H), 7.52 (d, J ) 7.5
Hz, 1H), 7.15 (m, 1H), 7.11 (d, J ) 8.6 Hz, 1H), 5.84 (m, 1H),
4.33 (m, 3H), 3.28 (dd, J ) 16.7, 5.5 Hz, 1H), 3.13 (dd, J )
16.5, 7.3 Hz, 1H). ES-MS m/z: 663 (M + H)+. HRMS (TOF):
exact mass calcd for C27H21N4O10SCl2 (M + H)+, 663.0349;
found, 663.0381.
6-[(4-Hydr oxy-3-m eth yl-ben zen esu lfon ylam in o)m eth yl]-
N-[1-(7-m et h oxy-b en zooxa zole-2-ca r b on yl)p r op yl]n ico-
tin a m id e (61). The advanced nicotinate intermediate was
prepared in the same manner as 40 except using 6-methylni-
cotinic acid as a reagent instead of 36. Compound 61 was
prepared using this advanced nicotinate intermediate and
42a 32 in the same manner as described for 42b. ES-MS m/z:
599 (M + H)+. HRMS (TOF): exact mass calcd for C26H23N4O11S
(M + H)+, 599.1089; found, 599.1078.
8.3 Hz, 1H), 8.32 (s, 1H), 7.92 (d, J ) 8.7 Hz, 1H), 7.78 (d, J
) 2.4 Hz, 1H), 7.10 (m, 1H), 4.70-4.73 (m, 1H), 4.69 (m, 1H),
4.34 (s, 2H), 2.75-2.82 (m, 1H), 2.60-2.68 (m, 1H). EI-MS
m/z: 452.1 (M + H)+. HRMS (TOF): exact mass calcd for
C
18H18N3O9S (M + H)+, 452.0758; found, 452.0778.
(S)-3-{[6-(Ben zen esu lfon yla m in o-m et h yl)p yr id in e-3-
ca r bon yl]a m in o}-4-oxo-bu tyr ic Acid Tr iflu or oa ceta te
(48). Compound 48 was prepared from resin 44e (8.4 mg, 28%).
1H NMR (CD3OD): δ 8.90 (s, 1H), 8.30 (dt, J ) 8.2, 2.1 Hz,
1H), 7.80-7.90 (m, 2H), 7.59-7.67 (m, 2H), 7.5-7.6 (m, 2H),
4.70 (d, J ) 11.1, 4.2 Hz, 1H), 4.47-4.56 (m, 1H), 4.31 (s, 2H),
2.74-2.84 (m, 1H), 2.60-2.70 (m, 1H).
(S)-3-({6-[(4-Hyd r oxy-ben zen esu lfon yla m in o)m eth yl]-
p yr id in e-3-ca r bon yl}a m in o)-4-oxo-bu tyr ic Acid Tr iflu o-
r oa ceta te (49). Compound 49 was prepared from resin 44e
(9.3 mg, 30%). 1H NMR (CD3OD): δ 8.90 (s, 1H), 8.30 (dt, J )
8.3, 2.0 Hz, 1H), 7.67 (m, 2H), 7.60 (d, J ) 8.2 Hz, 1H), (m,
2H), 4.70 (dd, J ) 11.1, 4.1 Hz, 1H), 4.46-4.56 (m, 1H), 4.23
(s, 2H), 2.60-2.70 (m, 1H), 2.74-2.84 (m, 2H).
(S )-3-{[5-(1-Ca r b oxym e t h yl-2-oxo-e t h ylca r b a m oyl)-
p yr id in -2-ylm eth yl]su lfa m oyl}ben zoic Acid Tr iflu or o-
a ceta te (50). Compound 50 was prepared from resin 44e (3.6
mg, 11%). 1H NMR (CD3OD): δ 8.84 (s, 1H), 8.44 (s, 1H), 8.15-
8.24 (m, 2H), 8.05 (d, J ) 7.8 Hz, 1H), 7.65 (t, J ) 7.9 Hz,
1H), 7.58 (d, J ) 8.3 Hz, 1H), 4.70 (dd, J ) 11.3, 4.1 Hz, 1H),
4.47-4.56 (m, 1H), 4.34 (s, 2H), 2.74-2.84 (m, 1H), 2.60-2.70
(m, 1H).
(S)-3-({6-[(3-Meth a n esu lfon yl-ben zen esu lfon yla m in o)-
m eth yl]p yr id in e-3-ca r bon yl}a m in o)-4-oxo-bu tyr ic Acid
Tr iflu or oa ceta te (51). Compound 51 was prepared from
resin 44e (6.7 mg, 19%). 1H NMR (CD3OD): δ 8.82 (s, 1H),
8.30 (s, 1H), 8.06-8.20 (m, 3H), 7.80 (t, J ) 7.9 Hz, 1H), 7.50
(d, J ) 8.1 Hz, 1H), 4.70 (dd, J ) 10.6, 4.1 Hz, 1H), 4.46-4.56
(m, 1H), 4.35 (s, 2H), 3.15 (s, 3H), 2.73-2.84 (m, 1H), 2.60-
2.70 (m, 1H). EI-MS m/z: 470.0 (M + H)+.
(S)-3-({6-[(4-Met h a n esu lfon yla m in o-b en zen esu lfon -
ylamino)methyl]pyridine-3-carbonyl}amino)-4-oxo-butyr ic
Acid Tr iflu or oa ceta te (52). Compound 52 was prepared
from resin 44e and 5 (10.7 mg, 30%). 1H NMR (CD3OD): δ
8.86 (s, 1H), 8.20 (dt, J ) 8.3, 1.7 Hz, 1H), 7.90 (d, J ) 8.6 Hz,
2H), 7.60 (d, J ) 8.2 Hz, 1H), 7.30 (d, J ) 8.4 Hz, 2H), 4.70
(dd, J ) 11.4, 4.0 Hz, 1H), 4.46-4.56 (m, 1H), 4.30 (s, 2H),
3.00 (s, 3H), 2.74-2.83 (m, 1H), 2.59-2.70 (m, 1H).
(S)-3-({6-[(3-Met h a n esu lfon yla m in o-b en zen esu lfon -
ylamino)methyl]pyridine-3-carbonyl}amino)-4-oxo-butyric
Acid Tr iflu or oa ceta te (53). Compound 53 was prepared
from resin 44e and 6 (5.8 mg, 16%). 1H NMR (CD3OD): δ 8.89
(s, 1H), 8.30 (dt, J ) 8.3, 1.8 Hz, 1H), 7.70 (s, 1H), 7.65 (d, J
) 8.4 Hz, 1H), 7.57 (m, 1H), 7.50 (t, J ) 7.8 Hz, 1H), 7.41 (m,
1H), 4.70 (dd, J ) 11.4, 4.1 Hz, 1H), 4.47-4.57 (m, 1H), 4.35
(s, 2H), 2.96 (s, 3H), 2.73-2.83 (m, 1H), 2.59-2.69 (m, 1H).
(S)-3-({6-[(3-Meth a n esu lfon yla m in oca r bon yl-ben zen e-
su lfon yla m in o)m et h yl]-p yr id in e-3-ca r b on yl}a m in o)-4-
oxo-bu tyr ic Acid Tr iflu or oa ceta te (54). Compound 54 was
prepared from resin 44e and 7 (14.0 mg, 37%). 1H NMR (CD3-
OD): δ 8.90 (s, 1H), 8.30 (m, 2H), 8.00-8.13 (m, 2H), 7.59-
7.72 (m, 2H), 4.70 (dd, J ) 11.4, 4.1 Hz, 1H), 4.47-4.56 (m,
1H), 4.40 (s, 2H), 3.40 (s, 3H), 2.74-2.83 (m, 1H), 2.60-2.70
(m, 1H). EI-MS m/z: 513.0 (M + H)+.
P r ep a r a tion of Resin 44a -e. Resin 4320 (63 g, 0.51 mmol/
g) was suspended in THF (150 mL) followed by addition of
acetic acid (1.0 mL, 17.5 mmol) and the appropriate ketone
(48.4 mmol). After it was stirred for 12 h, the resin was filtered,
washed successively with CH2Cl2 (3 × 200 mL) and ether (3
× 200 mL), and dried in vacuo to provide resin 44a -d . Resin
44e was prepared by literature procedures.19
Gen er a l Meth od for Solid -P h a se Syn th esis of Ald e-
h yd e a n d Keton e An a logu es. Resin 44a -e (300 mg, 0.06
mmol) was suspended in 20% piperidine in DMF (3 mL)
followed by gentle agitation for 20 min. After it was filtered,
the resin was washed successively with DMF (3 × 5 mL) and
CH2Cl2 (3 × 5 mL). The resin was then resuspended in DMF
(3 mL) followed by treatment with DIPEA (63 µL, 0.36 mmol),
the desired N-Fmoc-protected heterocyclic acid (0.12 mmol),
and PyBOP (93 mg, 0.18 mmol). After it was agitated at room
temperature for 12 h, the solution was drained and the resin
was washed successively with DMF (3 × 5 mL) and CH2Cl2 (3
× 5 mL). This resin was then treated with 20% piperidine in
DMF (3 mL) and agitated for 20 min. The resin was drained
and washed successively with DMF (3 × 5 mL) and CH2Cl2 (3
× 5 mL). To the resin were then added CH2Cl2 (3 mL), the
desired benzene sulfonyl chloride (0.12 mmol), and DIPEA
(0.063 mL, 0.36 mmol). The suspension was agitated at room
temperature for 12 h followed by filtration and washing with
DMF (3 × 5 mL) and CH2Cl2 (3 × 5 mL). The resin was then
cleaved under the appropriate cleavage conditions to provide
the fully functionalized analogue 45.
Gen er a l Meth od s for Clea va ge of Ald eh yd e An a logu es
fr om Resin . To derivatized aldehyde resin (300 mg, 0.06
mmol) was added THF/CH3CHO/HOAc/TFA (5:1:1:0.250, 3
mL). After it was agitated at room temperature for 3 h, the
resin was filtered and washed with CH2Cl2 (2 × 2 mL). The
filtrate and washings were combined, and the solvent was
removed under reduced pressure to provide a residue, which
was treated with CH2Cl2/H2O/TFA (1:1:0.1, 2 mL) at room
temperature for 30 min. The solvent was removed under
reduced pressure, and the product was purified by preparative
reverse-phase HPLC.
(S)-3-({6-[(3-Acetylsu lfa m oyl-ben zen esu lfon yla m in o)-
m eth yl]p yr id in e-3-ca r bon yl}a m in o)-4-oxo-bu tyr ic Acid
Tr iflu or oa ceta te (55). Compound 55 was prepared from
1
Gen er a l Meth od s for Clea va ge of Keton e An a logu es
fr om Resin . To derivatized ketone resin (300 mg, 0.06 mmol)
was added TFA/H2O (9:1, 3 mL). After it was agitated at room
temperature for 15 min, the resin was filtered and washed
with CH2Cl2 (2 × 2 mL). The filtrate and washings were
combined, and the solvent was removed under reduced pres-
sure to provide a residue, which was purified by preparative
reverse-phase HPLC.
resin 44e and 8 (10.0 mg, 27%). H NMR (CD3OD): δ 8.80 (s,
1H), 8.40 (m, 1H), 8.25 (dt, J ) 8.2, 2.0 Hz, 1H), 8.17 (d, J )
7.9 Hz, 1H), 8.05 (d, J ) 7.9 Hz, 1H), 7.72 (t, J ) 7.9 Hz, 1H),
7.58 (dd, J ) 8.2, 2.6 Hz, 1H), 4.70 (dd, J ) 10.1, 4.0 Hz, 1H),
4.47-4.56 (m, 1H), 4.38 (s, 2H), 2.73-2.83 (m, 1H), 2.59-2.69
(m, 1H), 2.0 (s, 3H). EI-MS m/z: 513.0 (M + H)+. HRMS
(TOF): exact mass calcd for C19H21N4O9S2 (M + H)+, 513.0744;
found, 513.0753.
(S)-5-{[5-(1-Ca r boxym eth yl-2-oxo-eth ylca r ba m oyl)p y-
(S)-4-Oxo-3-[(6-{[3-(1H -t e t r a zol-5-yl)b e n ze n e su lfon -

Small Molecule Inhibitors of Caspase-3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5019
ylam in o]m eth yl}pyr idin e-3-car bon yl)am in o]bu tyr ic Acid
Tr iflu or oa ceta te (56). Compound 56 was prepared from
resin 44e and 9 (8.7 mg, 25%). 1H NMR (CD3OD) δ 8.87 (s,
1H), 8.5 (s, 1H), 8.20-8.35 (m, 2H), 8.05 (d, J ) 7.8 Hz, 1H),
7.77 (t, J ) 7.8 Hz, 1H), 7.68 (d, J ) 8.1 Hz, 1H), 4.70 (dd, J
) 12.0, 4.1 Hz, 1H), 4.45-4.55 (m, 1H), 4.40 (s, 2H), 2.73-
2.83 (m, 1H), 2.59-2.69 (m, 1H). EI-MS m/z: 460.0 (M + H)+.
HRMS (TOF): exact mass calcd for C18H18N7O6S (M + H)+,
460.1033; found, 460.1037.
(S)-4-Oxo-3-[(6-{[3-(5-oxo-4,5-d ih yd r o[1,2,4]oxa d ia zol-
3-yl)b en zen esu lfon yla m in o]m et h yl}p yr id in e-3-ca r b on -
yl)a m in o]bu tyr ic Acid Tr iflu or oa ceta te (57). Compound
57 was prepared from resin 44e and 12 (11.0 mg, 31%). 1H
NMR (CD3OD): δ 8.87 (s, 1H), 8.18-8.30 (m, 2H), 7.95-8.10
(m, 2H), 7.73 (t d, J ) 7.9, 1.7 Hz, 1H), 7.61 (dd, J ) 8.2, 3.5
Hz, 1H), 4.70 (dd, J ) 11.4, 4.0 Hz, 1H), 4.47-4.57 (m, 1H),
4.37 (s, 2H), 2.74-2.83 (m, 1H), 2.59-2.69 (m, 1H). EI-MS
m/z: 476.0 (M + H)+.
3.2 Hz, 1H), 6.84 (d, J ) 2.6 Hz, 1H), 4.65 (dd, J ) 11.3, 4.1
Hz, 1H), 4.41 (m, 1H), 4.27 (d, J ) 1.3 Hz, 2H), 2.60-2.77 (m,
2H). EI-MS m/z: 457.0 (M + H)+. HRMS (TOF): exact mass
calcd for C16H16N3O9S2 (M + H)+, 458.0322; found, 458.0330.
(S)-5-({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-p r op ylca r ba m oyl]th ia zol-2-ylm eth yl}su lfa m oyl)-2-
h yd r oxy-ben zoic Acid (64b). Compound 64b was prepared
1
from resin 44a (1.7 mg, 5%). H NMR (CD3OD): δ 8.31 (d, J
) 2.6 Hz, 1H), 8.14 (s, 1H), 7.90 (d, J ) 6.7 Hz, 1H), 7.36-
7.37 (m, 2H), 7.21-7.23 (m, 2H), 7.06 (d, J ) 8.8 Hz, 1H), 5.12
(t, J ) 8.0 Hz, 1H), 4.40 (d, J ) 6.0 Hz, 2H), 3.92 (s, 2H), 3.41-
3.49 (m, 2H), 2.72-2.99 (m, 2H). EI-MS m/z: 628.0 (M + H)+.
(S)-5-{[3-(1-Ca r b oxym et h yl-2-oxo-et h ylca r b a m oyl)is-
oxa zol-5-ylm et h yl]su lfa m oyl}-2-h yd r oxy-b en zoic Acid
(65a ). Compound 65a was prepared from resin 44e (3.1 mg,
1
12%). H NMR (CD3OD): δ 8.28 (t, J ) 2.7 Hz, 1H), 7.85 (dt,
J ) 8.2, 2.0 Hz, 1H), 7.02 (dd, J ) 8.8, 2.8 Hz, 1H), 6.48 (d, J
) 4.4 Hz, 1H), 4.65 (dd, J ) 7.6, 4.3 Hz, 1H), 4.39-4.48 (m,
1H), 4.30 (s, 2H), 2.55-2.76 (m, 2H). HRMS (TOF): exact mass
calcd for C24H23N3O10S2Cl (M + H)+, 612.0507; found, 612.0535.
(S)-5-{[5-(1-Ca r b oxym et h yl-3-m et h ylsu lfa n yl-2-oxo-
p r op ylca r b a m oyl)p yr id in -2-ylm et h yl]su lfa m oyl}-2-h y-
1
d r oxy-ben zoic Acid (58). (4.3 mg, 14%). H NMR (CD3OD):
(S)-5-({3-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-p r op ylca r ba m oyl]isoxa zol-5-ylm eth yl}su lfa m oyl)-
2-h yd r oxy-ben zoic Acid (65b). Compound 65b was prepared
δ 8.88 (d, J ) 1.7 Hz, 1H), 8.23-8.27 (m, 2H), 7.88 (dd, J )
8.8, 2.4 Hz, 1H), 7.59 (d, J ) 8.2 Hz, 1H), 7.04 (J ) 8.8 Hz,
1H), 5.20 (t, J ) 6.4 Hz, 1H), 4.30 (s, 3H), 3.51 (d, J ) 14.7
Hz, 1H), 3.40 (d, J ) 14.7 Hz, 1H), 3.03 (dd, J ) 16.9, 6.2 Hz,
1H), 2.80 (dd, J ) 16.9, 6.9 Hz, 1H), 2.66 (s, 2H). EI-MS m/z:
512 (M + H)+.
1
from resin 44a (8.5 mg, 23%). H NMR (CD3OD): δ 8.25 (d, J
) 2.4 Hz, 1H), 7.85 (dd, J ) 8.8, 2.4 Hz, 1H), 7.30-7.40 (m,
2H), 7.15-7.25 (m, 2H), 7.00 (d, J ) 8.8 Hz, 1H), 6.51 (s, 1H),
5.15 (t, J ) 6.0 Hz, 1H), 4.30 (s, 2H), 3.79 (d, J ) 3.1 Hz, 2H),
3.40 (q, J ) 15.3 Hz, 1H), 2.70-3.05 (m, 2H). EI-MS m/ z:
612.0 (M + H)+.
(S)-5-({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-p r op ylca r ba m oyl]p yr id in -2-ylm eth yl}su lfa m oyl)-
2-h yd r oxy-ben zoic Acid (59). Compound 59 was prepared
from resin 44a (14.4 mg, 33%). 1H NMR (CDCl3): δ 8.86 (s,
1H), 8.27 (s, 1H), 8.20 (d, J ) 6.1 Hz, 1H), 7.88 (d, J ) 6.9 Hz,
1H), 7.56 (d, J ) 7.6 Hz, 1H), 7.22-7.40 (m, 4H), 7.03-7.05
(m, 1H), 5.18 (m, 1H), 4.29 (s, 2H), 3.83 (s, 2H), 3.44-3.48 (m,
2H), 2.81-3.02 (m, 2H). EI-MS m/z: 622.0 (M + H)+. HRMS
(S )-5-{[5-(1-Ca r b oxym e t h yl-2-oxo-e t h ylca r b a m oyl)-
th iop h en -2-ylm eth yl]su lfa m oyl}-2-h yd r oxy-ben zoic Acid
(66a ). Compound 66a was prepared from resin 44e (3.2 mg,
1
12%). H NMR (CD3OD): δ 8.23 (s, 1H), 7.80 (d, J ) 8.8 Hz,
1H), 7.39 (t, J ) 3.9 Hz, 1H), 6.97 (dd, J ) 8.7, 2.6 Hz, 1H),
6.78 (d, J ) 3.6 Hz, 1H), 4.58 (dd, J ) 11.2, 4.1 Hz, 1H), 4.35-
4.37 (m, 1H), 4.23 (s, 2H), 2.58-2.81 (m, 2H). EI-MS m/z: 457.0
(M + H)+. HRMS (TOF): exact mass calcd for C17H17N2O9S2
(M + H)+, 457.0370; found, 457.0386.
(TOF): exact mass calcd for
622.0715; found, 622.0719.
C
26H25N3O9S2Cl (M + H)+,
(S )-5-{[5-(1-Ca r b oxym e t h yl-2-oxo-e t h ylca r b a m oyl)-
pyr im idin -2-ylm eth yl]su lfam oyl}-2-h ydr oxy-ben zoic Acid
(62a ). Compound 62a was prepared from resin 44e (1.1 mg,
(S)-5-({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-pr opylcar bam oyl]th ioph en -2-ylm eth yl}su lfam oyl)-
2-h yd r oxy-ben zoic Acid (66b). Compound 66b was prepared
1
4%). H NMR (CDCl3): δ 8.95 (d, J ) 2.8 Hz, 2H), 8.25 (dd, J
) 6.3, 2.4 Hz, 1H), 7.84-7.88 (m, 1H), 6.99 (dd, J ) 8.8, 3.9
Hz, 1H), 4.68 (dd, J ) 9.1, 4.0 Hz, 1H), 4.47-4.51 (m, 1H),
4.40 (d, J ) 1.7 Hz, 2H), 2.62-2.77 (m, 2H). EI-MS m/z: 453.0
(M + H)+. HRMS (TOF): exact mass calcd for C17H17N4O9S
(M + H)+, 453.0710; found, 453.0700.
1
from resin 44a (5.1 mg, 14%). H NMR (CD3OD): δ 8.26 (d, J
) 2.2 Hz, 1H), 7.85 (dd, J ) 8.8, 2.3 Hz, 1H), 7.45 (d, J ) 3.7
Hz, 1H), 7.33-7.37 (m, 2H), 7.16-7.22 (m, 2H), 7.00 (d, J )
8.8 Hz, 1H), 6.86 (d, J ) 3.6 Hz, 1H), 5.09 (t, J ) 6.3 Hz, 1H),
4.30 (s, 2H), 3.78 (d, J ) 3.9 Hz, 2H), 3.44 (d, J ) 15.3 Hz,
1H), 3.36 (d, J ) 15.3, 1H), 3.00 (dd, J ) 16.9, 6.5 Hz, 1H),
2.75 (dd, J ) 16.8, 6.5 Hz, 1H). EI-MS m/z: 627.0 (M + H)+.
Anal. Calcd for C25H23N2O9S3Cl: C, 47.88; H, 3.70; N, 4.23.
Found: C, 47.67; H, 3.61; N, 4.23.
(S)-5-({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-pr opylcar bam oyl]pyr im idin -2-ylm eth yl}su lfam oyl)-
2-h yd r oxy-ben zoic Acid (62b). Compound 62b was prepared
from resin 44a (6.4 mg, 17%). EI-MS m/z: 623.0 (M + H)+.
HRMS (TOF): exact mass calcd for C25H24N4O9S2Cl (M + H)+,
623.0667; found, 623.0685.
(S )-5-{[5-(1-Ca r b oxym e t h yl-2-oxo-e t h ylca r b a m oyl)-
p yr a zin -2-ylm eth yl]su lfa m oyl}-2-h yd r oxy-ben zoic Acid
(63a ). Compound 63a was prepared from resin 44e (3.2 mg,
12%). 1H NMR (CDCl3): δ 9.04 (s, 1H), 8.58 (d, J ) 4.9 Hz,
1H), 8.24 (m, 1H), 7.87 (d, J ) 8.8 Hz, 1H), 7.02 (d, J ) 6.9
Hz, 1H), 4.72 (t, J ) 5.4 Hz, 1H), 4.50 (m, 1H), 4.35 (s, 2H),
2.65-2.74 (m, 2H). EI-MS m/z: 453.0 (M + H)+.
(S )-5-{[4-(1-Ca r b oxym e t h yl-2-oxo-e t h ylca r b a m oyl)-
th iop h en -2-ylm eth yl]su lfa m oyl}-2-h yd r oxy-ben zoic Acid
(67a ). Compound 67a was prepared from resin 44e (15.6 mg,
57%). 1H NMR (CD3OD): δ 8.28 (d, J ) 2.3 Hz, 1H), 7.84-
7.88 (m, 2H), 7.23 (d, J ) 5.1 Hz, 1H), 7.03 (dd, J ) 8.8, 2.4
Hz, 1H), 4.66 (dd, J ) 13.3, 4.0 Hz, 1H), 4.40-4.47 (m, 1H),
4.26 (s, 2H), 2.69-2.77 (m, 1H), 2.57-2.64 (m, 1H). EI-MS
m/z: 457.0 (M + H)+.
(S)-5-({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-pr opylcar bam oyl]th ioph en -3-ylm eth yl}su lfam oyl)-
2-h yd r oxy-ben zoic Acid (67b). Compound 67b was prepared
(S)-5-({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-p r op ylca r ba m oyl]p yr a zin -2-ylm eth yl}su lfa m oyl)-
2-h yd r oxy-ben zoic Acid (63b). Compound 63b was prepared
1
1
from resin 44a (5.2 mg, 14%). H NMR (CD3OD): δ 8.27 (d, J
from resin 44a (0.8 mg, 2%). H NMR (CDCl3): δ 7.45 (d, J )
) 2.3 Hz, 1H), 7.91 (d, J ) 1.2 Hz, 1H), 7.85 (dd, J ) 8.8, 2.4
Hz, 1H), 7.34-7.38 (m, 2H), 7.18-7.23 (m, 3H), 7.01 (d, J )
8.8 Hz, 1H), 5.09 (t, J ) 6.4 Hz, 1H), 4.28 (s, 2H), 3.81 (d, J )
3.6 Hz, 2H), 3.44 (d, J ) 15.2 Hz, 1H), 3.36 (d, J ) 15.2 Hz,
1H), 3.00 (dd, J ) 16.8, 6.4 Hz, 1H), 2.75 (dd, J ) 16.8, 6.5
Hz, 1H). EI-MS m/z: 627.0 (M + H)+.
1.3 Hz, 1H), 6.99 (s, 1H), 6.63 (d, J ) 1H), 6.26 (dd, J ) 8.8,
2.4 Hz, 1H), 5.74-5.80 (m, 2H), 5.60-5.63 (m, 2H), 5.42 (d, J
) 8.8 Hz, IH), 3.62-3.63 (m, 1H), 2.78 (s, 2H), 2.22 (s, 2H),
1.89 (d, J ) 15.4 Hz, 1H), 1.85 (d, J ) 15.1 Hz, 1H), 1.41 (dd,
J ) 16.8, 5.6 Hz, 1H), 1.33 (dd, J ) 16.8, 5.5 Hz, 1H). EI-MS
m/z: 623.0 (M + H)+.
(S )-5-{[5-(1-Ca r b oxym e t h yl-2-oxo-e t h ylca r b a m oyl)-
t h ia zol-2-ylm et h yl]su lfa m oyl}-2-h yd r oxy-b en zoic Acid
(64a ). Compound 64a was prepared from resin 44e (5.5 mg,
20%). 1H NMR (CD3OD): δ 8.28 (d, J ) 2.6 Hz, 1H), 7.85-
7.88 (m, 1H), 7.45 (dd, J ) 5.6, 3.9 Hz, 1H), 7.03 (dd, J ) 8.9,
(S )-5-{[5-(1-Ca r b oxym e t h yl-2-oxo-e t h ylca r b a m oyl)-
th iop h en -3-ylm eth yl]su lfa m oyl}-2-h yd r oxy-ben zoic Acid
(68a ). Compound 68a was prepared from resin 44e (17.6 mg,
64%). H NMR (CD3OD): δ 8.26 (s, 1H), 7.86 (d, J ) 8.8 Hz,
1H), 7.51 (d, J ) 7.0 Hz, 1H), 7.34 (s, 1H), 7.04 (dd, J ) 8.8,
1

5020 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Choong et al.
2.6 Hz, 1H), 4.67 (dd, J ) 12.4, 4.1 Hz, 1H), 4.43-4.44 (m,
1H), 4.06 (s, 2H), 2.64-2.72 (m, 2H). EI-MS m/z: 457.0 (M +
H)+.
(dd, J ) 8.8, 2.4 Hz, 1H), 7.44 (m, 1H), 7.20 (m, 2H), 7.13 (m,
3H), 7.02 (d, J ) 8.9 Hz, 1H), 6.88 (d, J ) 3.81 Hz, 1H), 4.80
(t, J ) 6.5 Hz, 1H), 4.31 (s, 2H), 2.96 (dd, J ) 16.8, 6.6 Hz,
1H), 2.67 (m, 1H), 2.58 (m, 4H), 1.89 (m, 2H).
(S)-4-[(4-H yd r oxy-3-m et h yl-b en zen esu lfon yla m in o)-
m eth yl]th iop h en e-2-ca r boxylic Acid [3-(2-Ch lor o-ben z-
ylsu lfa n yl)-1-eth yl-2-oxo-p r op yl]a m id e (68b). Compound
68a was prepared from resin 44a (4.5 mg, 12%). 1H NMR (CD3-
OD): δ 8.28 (d, J ) 2.3 Hz, 1H), 7.84 (dd, J ) 8.8 2.4 Hz, 1H),
7.47 (s, 1H), 7.34-7.38 (m, 2H), 7.20-7.23 (m, 2H), 6.99 (d, J
) 8.9 Hz, 1H), 5.09 (t, J ) 6.3 Hz, 1H), 4.26 (s, 2H), 3.81 (s,
2H), 3.42-3.47 (m, 2H), 2.99 (dd, J ) 16.9, 6.4 Hz, 1H), 2.73
(dd, J ) 16.7, 6.5 Hz, 1H). EI-MS m/z: 707.0 (M + 79)+.
(S)-5-{[5-(1-Ca r b oxym et h yl-2-oxo-et h ylca r b a m oyl)-4-
m eth yl-th iop h en -2-ylm eth yl]su lfa m oyl}-2-h yd r oxy-ben -
zoic Acid (69a ). Compound 69a was prepared from resin 44e
(S)-3-{[5-(Ben zen esu lfon yla m in o-m eth yl)th iop h en e-2-
ca r bon yl]a m in o}-5-(2-ch lor o-ben zylsu lfa n yl)-4-oxo-p en -
ta n oic Acid (74). Compound 74 was prepared from resin 44a
(10.8 mg, 32%). 1H NMR (CD3OD): δ 7.79 (d, J ) 7.4 Hz, 2H),
7.55 (d, J ) 7.3 Hz, 1H), 7.49 (d, J ) 7.7 Hz, 2H), 7.46 (m,
1H), 7.34 (m, 2H), 7.21 (m, 2H), 6.86 (d, J ) 3.8 Hz, 1H), 5.08
(t, J ) 6.5 Hz, 1H), 3.82 (d, J ) 13.3 Hz, 1H), 3.77 (d, J ) 13.3
Hz, 1H), 3.39 (m, 2H), 2.99 (dd, J ) 16.9, 6.3 Hz, 1H), 2.74
(dd, J ) 16.8, 6.8 Hz, 1H). EI-MS m/z: 567.0 (M + H)+.
(S)-5-(2-Ch lor o-ben zylsu lfa n yl)-3-({5-[(4-h yd r oxy-ben -
zen esu lfon ylam in o)m eth yl]th ioph en e-2-car bon yl}am in o)-
4-oxo-p en ta n oic Acid (75). Compound 75 was prepared from
resin 44a . 1H NMR (CD3OD): δ 7.62 (m, 2H), 7.45 (d, J ) 3.81
Hz, 1H), 7.32 (m, 2H), 7.16 (m, 2H), 6.83 (m, 3H), 5.05 (t, J )
6.6 Hz, 1H), 4.19 (s, 2H), 3.76 (m, 2H), 3.37 (m, 2H), 3.26 (m,
2H), 2.96 (dd, J ) 16.8, 6.4 Hz, 1H), 2.70 (dd, J ) 16.8, 6.6
Hz, 1H).
1
(11.9 mg, 42%). H NMR (CD3OD): δ 8.24 (s, 1H), 7.84 (dd, J
) 8.8, 1.0 Hz, 1H), 7.03 (d, J ) 8.8 Hz, 1H), 6.62 (s, 1H), 4.68
(dd, J ) 9.9, 4.1 Hz, 1H), 4.37 (m, 1H), 4.23 (s, 2H), 2.65-2.68
(m, 2H), 2.31 (s, 3H). EI-MS m/z: 471.0 (M + H)+. HRMS
(TOF): exact mass calcd for C18H19N2O9S2 (M + H)+, 471.0526;
found, 471.0544.
(S)-5-({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-p r op ylca r ba m oyl]-4-m eth yl-th iop h en -2-ylm eth yl}-
su lfa m oyl)-2-h yd r oxy-ben zoic Acid (69b). Compound 69b
was prepared from resin 44a (3.8 mg, 10%). 1H NMR (CD3-
OD): δ 6.70 (s, 1H), 6.30 (dd, J ) 8.8, 2.2 Hz, 1H), 5.82-5.87
(m, 2H), 5.68 (m, 2H), 5.48 (d, J ) 8.8 Hz, 1H), 5.13 (s, 1H),
3.51 (t, Hz, 1H), 1.77 (s, 3H), 1.44 (dd, J ) 16.9, 5.8 Hz, 1H)),
1.29 (dd, J ) 16.8, 5.8 Hz, 1H). EI-MS m/z: 641.0 (M + H)+.
HRMS (TOF): exact mass calcd for C26H26N2O9S3Cl (M + H)+,
641.0483; found, 641.0490.
(S)-3-({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-pr opylcar bam oyl]th ioph en -2-ylm eth yl}su lfam oyl)-
ben zoic Acid (76). Compound 76 was prepared from resin
1
44a (4.3 mg, 12%). H NMR (CD3OD): δ 8.37 (s, 1H), 8.16 (d,
J ) 7.8 Hz, 1H), 7.97 (d, J ) 7.9 Hz, 1H), 7.59 (t, J ) 7.8 1H),
7.42 (d, J ) 3.8 Hz, 1H), 7.36 (m, 1H), 7.21 (m, 1H), 6.86 (d,
3.8 Hz, 1H), 5.08 (t, J ) 6.6 Hz, 1H), 4.33 (s, 2H), 3.80 (d, J )
4.3 Hz, 2H), 3.44 (d, J ) 15.2 Hz, 1H), 3.35 (d, J ) 15.3 Hz,
1H), 3.00 (dd, J ) 16.9, 6.5 Hz, 1H), 2.74 (dd, J ) 16.8, 6.7
Hz, 1H). EI-MS m/z: 611.0 (M + H)+.
(S )-5-{[5-(1-Ca r b oxym e t h yl-2-oxo-e t h ylca r b a m oyl)-
fu r a n -2-ylm et h yl]su lfa m oyl}-2-h yd r oxy-b en zoic Acid
(70a ). Compound 70a was prepared from resin 44e (2.5 mg,
9%). 1H NMR (CD3OD): δ 8.23 (s, 1H), 7.83 (d, J ) 9.1 Hz,
1H), 7.02 (d, J ) 8.8 Hz, 1H), 6.91 (s, 1H), 6.22 (s, 1H), 4.68-
4.74 (m, 1H), 4.43-4.49 (m, 1H), 4.21 (s, 2H), 2.60-2.74 (m,
2H). EI-MS m/z: 441.0 (M + H)+.
(S)-5-(2-Ch lor o-ben zylsu lfa n yl)-4-oxo-3-[(5-{[3-(5-oxo-
4,5-d ih yd r o[1,2,4]oxa d ia zol-3-yl)ben zen esu lfon yla m in o]-
m eth yl}th ioph en e-2-car bon yl)am in o]pen tan oic Acid (77).
Compound 77 was prepared from resin 44a and 11 (3.4 mg,
9%). 1H NMR (CD3OD): δ 8.16 (s, 1H), 7.98 (d, J ) 8.2 Hz,
1H), 7.95 (d, J ) 7.9 Hz, 1H), 7.66 (t, J ) 7.9 Hz, 1H), 7.42 (d,
J ) 3.7 Hz, 1H), 7.34-7.38 (m, 2H), 7.20-7.23 (m, 2H), 6.88
(d, J ) 3.6 Hz, 1H), 5.08 (t, J ) 6.4 Hz, 1H), 4.38 (s, 2H), 3.80
(d, J ) 3.7 Hz, 2H), 3.44 (d, J ) 15.2 Hz, 1H), 3.37 (d, J )
15.3 Hz, 1H), 2.99 (dd, J ) 16.8, 6.4 Hz, 1H), 2.75 (dd, J )
16.9, 6.6 Hz, 1H). EI-MS m/z: 651.0 (M + H)+.
(S)-5-({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-p r op ylca r ba m oyl]fu r a n -2-ylm eth yl}su lfa m oyl)-2-
h yd r oxy-ben zoic Acid (70b). Compound 70b was prepared
1
from resin 44a (5.5 mg, 14%). H NMR (CD3OD): δ 8.20 (d, J
) 2.0 Hz, 1H), 7.82 (dd, J ) 8.8, 2.0 Hz, 1H), 7.33-7.38 (m,
2H), 7.19-7.21 (m, 2H), 6.99 (d, J ) 8.8 Hz, 1H), 6.92 (d, J )
3.4 Hz, 1H), 6.27 (d, J ) 3.3 Hz, 1H), 5.11 (t, J ) 6.2 Hz, 1H),
4.18 (s, 2H), 3.80 (d, J ) 2.8 Hz, 2H), 3.44 (d, J ) 15.2 Hz,
1H), 3.36 (d, J ) 15.3 Hz, 1H), 2.98 (dd, J ) 16.9, 6.2 Hz,
1H), 2.79 (d, J ) 16.9, 6.2 Hz, 1H). EI-MS m/z: 611.0 (M +
H)+.
(S)-5-({5-[1-Ca r boxym eth yl-2-oxo-3-(p yr id in -3-ylm eth -
y ls u lfa n y l)p r o p y lc a r b a m o y l]t h io p h e n -2-y lm e t h y l}-
su lfa m oyl)-2-h yd r oxy-ben zoic Acid (71). Compound 71
was prepared from resin 44c (7.3 mg, 20%). 1H NMR (CD3-
OD): δ 8.81 (br s, 1H), 8.69 (br s, 1H), 8.52 (br s, 1H), 8.27 (d,
J ) 2.0 Hz, 1H), 7.93 (br s, 1H), 7.85 (dd, J ) 8.8, 2.3 Hz, 1H),
7.45 (d, J ) 3.8 Hz, 1H), 7.02 (d, J ) 8.8 Hz, 1H), 6.89 (d, J )
3.7 Hz, 1H), 4.98 (m, 1H), 4.30 (s, 2H), 3.95 (s, 2H), 3.51 (d, J
) 15.3 Hz, 1H), 3.41 (d, J ) 15.3 Hz, 1H), 3.00 (dd, J ) 17.0,
7.0 Hz, 1H), 2.72 (dd, J ) 17.0, 5.9 Hz, 1H). EI-MS m/z: 594
(M + H)+.
(S)-5-({5-[1-Ca r boxym eth yl-2-oxo-3-(p yr id in -4-ylm eth -
y ls u lfa n y l)p r o p y lc a r b a m o y l]t h io p h e n -2-y lm e t h y l}-
su lfa m oyl)-2-h yd r oxy-ben zoic Acid (72). Compound 72
was prepared from resin 44d . 1H NMR (CD3OD): δ 8.71 (br s,
2H), 8.27 (d, J ) 2.2 Hz, 1H), 8.0 (br s, 2H), 7.85 (dd, J ) 8.8,
2.2 Hz, 1H), 7.46 (d, J ) 3.7 Hz, 1H), 7.02 (d, J ) 8.8 Hz, 1H),
6.89 (d, J ) 3.8 Hz, 1H), 4.98 (m, 1H), 4.30 (s, 2H), 3.99 (s,
2H), 3.52 (d, J ) 15.1 Hz, 1H), 3.43 (d, J ) 15.1 Hz, 1H), 3.00
(dd, J ) 16.7, 6.7 Hz, 1H), 2.72 (dd, J ) 16.7, 6.0 Hz, 1H).
EI-MS m/z: 594 (M + H)+.
(S)-5-({5-[1-Ca r boxym eth yl-5-(2-ch lor o-p h en yl)-2-oxo-
p en tylca r ba m oyl]th iop h en -2-ylm eth yl}su lfa m oyl)-2-h y-
d r oxy-ben zoic Acid (73). Compound 73 was prepared from
resin 44b. 1H NMR (CD3OD): δ 8.27 (d, J ) 2.5 Hz, 1H), 7.85
(S)-5-({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-pr opylcar bam oyl]th ioph en -2-ylm eth yl}m eth yl-su l-
fa m oyl)-2-h yd r oxy-ben zoic Acid (78). Compound 78 was
1
prepared from resin 44a (5.8 mg, 15%). H NMR (CD3OD): δ
8.28 (s, 1H), 7.89 (d, J ) 8.9 Hz, 1H), 7.55 (d, J ) 3 Hz, 1H),
7.34-7.36 (m, 2H), 7.19-7.21 (m, 2H), 7.11 (dd, J ) 8.8, 2.5
Hz, 1H), 7.01 (s, 1H), 5.10 (t, J ) 9.0 Hz, 1H), 4.44 (s, 2H),
3.80 (d, J ) 3.5 Hz, 2H), 3.46 (d, J ) 15.3 Hz, 1H), 3.38 (d, J
) 15.2 Hz, 1H), 2.85-3.04 (m, 1H), 2.76-2.78 (m, 1H), 2.72
(s, 3H). EI-MS m/z: 641.0 (M + H)+.
(S)-5-({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-p r op ylca r b a m oyl]t h iop h e n e -2-su lfon yla m in o}-
m eth yl)-2-h yd r oxy-ben zoic Acid (79). Compound 79 was
1
prepared from resin 44a (1.0 mg, 3%). H NMR (CD3OD): δ
7.71 (d, J ) 1.9 Hz, 1H), 7.60 (d, J ) 3.9 Hz, 1H), 7.45 (d, J )
3.9 Hz, 1H), 7.36 (m, 3H), 7.21 (m, 2H), 6.82 (d, J ) 8.5 Hz,
1H), 5.15 (t, J ) 6.5 Hz, 1H), 4.12 (s, 2H), 3.82 (s, 2H), 3.48 (d,
J ) 15.3 Hz, 1H), 3.40 (d, J ) 15.3 Hz, 1H), 3.16 (m, 1H),
2.86-3.01 (m, 1H), 2.75-2.85 (m, 1H). EI-MS m/z: 627.0 (M
+ H)+.
(S)-5-[({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-p r op ylca r b a m oyl]t h iop h en -2-ylm et h yl}a m in o)-
m eth yl]-2-h yd r oxy-ben zoic Acid (47a ). Compound 47a was
prepared from resin 44a (12.2 mg, 35%). 1H NMR (CD3OD):
δ 8.05 (m, 1H), 7.71 (m, 1H), 7.58 (m, 1H), 7.36 (m, 3H), 7.21
(m, 2H), 7.01 (m, 1H), 5.14-5.17 (m, 1H), 4.50 (s, 2H), 4.21 (s,
2H), 3.81 (s, 2H), 3.46 (d, J ) 15.6 Hz, 1H), 3.39 (d, J ) 15.1
Hz, 1H), 3.02 (dd, J ) 17.0, 6.3 Hz, 1H), 2.78 (J ) 17.0, 7.2
Hz, 1H). EI-MS m/z: 427.0 (M + H)+.
(S)-5-[(Acetyl-{5-[1-ca r boxym eth yl-3-(2-ch lor o-ben zyl-
su lfa n yl)-2-oxo-p r op ylca r ba m oyl]th iop h en -2-ylm eth yl}-
a m in o)m eth yl]-2-h yd r oxy-ben zoic Acid (47b). Compound

Small Molecule Inhibitors of Caspase-3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5021
47b was prepared from resin 44a (2.4 mg, 6%). 1H NMR (CD3-
OD; reported as a mixture of rotamers): δ 7.90 (s, 1H), 7.75
(s, 0.5H), 7.69 (d, 0.5H), 7.59 (d, J ) 3.6 Hz, 0.5H), 7.50 (d, J
) 3.9 Hz, 0.5H), 7.32-7.39 (m, 3H), 7.20-7.22 (m, 2H), 5.08-
5.11 (m, 1H), 4.66-4.73 (m, 1H), 3.80 (d, J ) 4.7 Hz, 2H), 3.46
(d, J ) 15.3 Hz, 1H), 3.38 (d, J ) 15.7 Hz, 1H), 2.99-3.04 (m,
1H), 2.72-2.78 (m, 1H), 2.66 (s, 3H), 2.21 (s, 1H). EI-MS m/z:
in complex with caspase-3. Initial molecular dynamics models
were built by placing the proposed compound in the protein
active site using crystal structure coordinates15 surrounded by
a 20 Å sphere of intact protein residues, plus sufficient TIP3P
waters to fill a rectangular box with a 5 Å buffer. Sodium and
chloride ions were introduced as needed to achieve neutrality.
Simulations were performed using the particle mesh Ewald
electrostatic approximation with an NTP ensemble, an inte-
gration step of 1 fs, and with shake constraints applied to
bonds involving hydrogen. Each simulation was initiated with
30 ps of solvent relaxation during which all solute atoms were
subjected to harmonic positional restraint with a force constant
of 1.0 (kcal/mol‚Å2). Thereafter, only solute atoms >15 Å from
the compound were restrained.
619.0 (M + H)+. HRMS (TOF): exact mass calcd for C28H28
-
N2O8S2Cl (M + H)+, 619.0970; found, 619.0996.
(S)-5-[({5-[1-Car boxym eth yl-3-(2-ch lor o-ben zylsu lfan yl)-
2-oxo-pr opylcar bam oyl]th ioph en -2-ylm eth yl}m eth yl-am i-
n o)m eth yl]-2-h yd r oxy-ben zoic Acid (47c). Compound 47c
was prepared from resin 44a (5.8 mg, 16%). 1H NMR (CD3-
OD): δ 8.06 (d, J ) 2.0 Hz, 1H), 7.74 (d, J ) 3.6 Hz, 1H), 7.61
(dd, J ) 8.3, 1.8 Hz, 1H), 7.35-7.42 (m, 3H), 7.21-7.23 (m,
2H), 7.05 (d, J ) 8.5 Hz, 1H), 5.49 (s, 1H), 5.16 (t, J ) 6.4 Hz,
1H), 4.71 (1H), 4.47-4.59 (m, 2H), 4.24-4.29 (m, 1H), 3.81
(d, J ) 2.2 Hz, 2H), 3.47 (d, J ) 15.2 Hz, 1H), 3.40 (d, J )
15.0 Hz, 1H), 3.03 (dd, J ) 16.7, 5.7 Hz, 1H), 2.75-2.83 (m,
1H). EI-MS m/z: 591.0 (M + H)+.
Cr ysta lliza tion of th e Com p lex of Ca sp a se-3 w ith
Com p ou n d s 3, 4, a n d 66b. Crystals of caspase-3 were grown
at 20 °C using the hanging drop vapor diffusion method. Equal
volumes of protein solution (5-10 mg/mL of previously modi-
fied protein in 10 mM Tris pH 8.5) were mixed with the
reservoir solution containing 100 mM sodium citrate, pH 5.9,
4% glycerol, 10-20% PEG6000, and 10 mM DTT. Crystalliza-
tion of caspase-3 and compound 3 required using the 2,6-
dichloroacyloxymethyl ketone of 3. Crystallization of caspase-3
and compound 4 required using the fluoromethyl ketone of 4.
For the complex between caspase-3 and compound 66b, a
2-fold excess of 66b was added to the protein solution prior to
setting up the drops.
En zym e Assa ys. The effectiveness of compounds against
the activity of human recombinant caspase-1-8 was measured
using fluorometric assays. Caspase-1, -2, and -4-7 were
purchased from BIOMOL Research Laboratories Inc. Caspase-3
and -8 were expressed and purified as described.8,41 Coumarin-
based fluorogenic substrates that incorporated optimal peptide
recognition motifs for each enzyme were purchased from Alexis
Biochemicals. Enzyme was added to test compounds titrated
in dimethyl sulfoxide (DMSO) and incubated at room temper-
ature for 30 min. Substrate addition initiated the reaction,
bringing the final reaction volume to 50 µL. Preferred 1X buffer
solutions were as follows: 25 mM HEPES, pH 7.4, 0.1%
CHAPS, 50 mM KCl, and 5 mM â-mercaptoethanol (â-ME) for
caspase-1-4 and -6; 100 mM HEPES, pH 7.4, 10% sucrose,
0.1% CHAPS, 200 mM NaCl, and 10 mM DTT for caspase-5;
25 mM HEPES, pH 7.4, 0.1% CHAPS, 50 mM KCl, 5 mM
CaCl2, and 5 mM â-ME for caspase-7; 100 mM HEPES, pH
7.4, 10% sucrose, 0.1% CHAPS, and 10 mM DTT for caspase-
8. The final concentration of the peptide substrates (and the
corresponding enzyme) was 4 µM Ac-Trp-Glu-His-Asp-AFC (3
nM caspase-1), 40 µM Ac-Val-Asp-Val-Ala-Asp-AFC (17 nM
caspase-2), 5 µM Ac-Asp-Glu-Val-Asp-AFC (1.6 nM caspase-
3), 30 µM Ac-Trp-Glu-His-Asp-AFC (60 nM caspase-4 and 83
nM caspase-5), 7 µM Ac-Val-Glu-Ile-Asp-AFC (2 nM caspase-
6), 30 µM Ac-Asp-Glu-Val-Asp-AFC (1 nM caspase-7), and 3
µM Ac-Ile-Glu-Thr-Asp-AFC (10 nM caspase-8). Assays were
carried out in black 96 well flat bottom, polystyrene plates
(Corning Incorporated Costar 3915). Caspase activity was
monitored using a Molecular Devices’ Microplate Spectrofluo-
rometer Gemini XS with an excitation wavelength of 365 nm
and an emission wavelength of 495 nm. Kinetic data were
collected over a 15 min assay run at room temperature. IC50
values were calculated through direct fits of the data to a four
parameter fit curve using the computer application SOFTmax
PRO. Ki(apparent) values were calculated using the following
equation: Ki(app) ) IC50/(1 + [substrate]/Km).
Small rhombic plates usually appeared after 1-2 weeks.
They reached their maximum size of approximately 200 µm
× 200 µm × 20 µm after 2 months. Before data collection,
crystals were dipped briefly into reservoir solution containing
25% glycerol and then flash-frozen in liquid nitrogen.
Diffraction data for caspase-3 modified with the 2,6-dichlo-
roacyloxymethyl ketone of 3 and the fluoromethyl ketone of 4
were collected at 100 K using a Rigaku (Tokyo) RU-3R
generator, an R-axis-IV detector, and processed using D*Trek.46
Diffraction data for caspase-3 modified with 66b were collected
at beamline 7.1 of the Stanford Synchrotron Radiation Labora-
tory and processed using programs Mosflm48 and Scala. The
crystals were isomorphous with previously solved structures;
therefore, refinement began with rigid body refinement using
Refmac.46 Compound models were constructed in Pymol,41 and
the models were adjusted using program O49 and refined using
program Refmac.50
Ack n ow led gm en t. We thank the Sunesis automa-
tion group (Stuart Lam, Thomas Webb, and Alex Hsi)
for their help in preparatory HPLC purifications and
Simone Evarts and Mark Cancilla for HRMS determi-
nation.
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Typically, all compounds with a Ki < 0.2 µM were screened
at least two times, and usually more, against caspase-3.
Because control compounds were included in all assays, we
have been able to estimate the accuracy of our in vitro assays.
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